Therapeutic use for alpha1 proteinase inhibitor in hematopoiesis

ABSTRACT

A previously unrecognized fundamental property of α 1 PI is to regulate the phenotypic composition of circulating and tissue-associated cells derived from hematopoietic stem cells. The present invention comprises screening for various unmodified and modified α 1 PI&#39;s which are useful in the treatment of abnormalities in the number of cells of myeloid or lymphoid lineage that are associated with HW-1 infection, microbial infection, leukemia, solid tumor cancers, atherosclerosis, autoimmunity, stem cell transplantation, organ transplantation, and other diseases affected by cells of the immune system. The interaction of α 1 PI with its receptors, HLE CS  and LRP, influences the level of cells of different lineages. Genetic and proteolytic modification of α 1 PI is used to target these receptors to increase or decrease specific cell populations, as needed, in the various disease states.

This application claims priority under 35 U.S.C. §119(e) fromProvisional Application No. 60/748,137 filed Dec. 6, 2005.

BACKGROUND OF THE INVENTION

Full length active α₁proteinase inhibitor (α₁PI, α₁antitripsin) iscomposed of 394 amino acids (aa) having a mass of approximately 55kDawhen fully glycosylated (Berninger, 1985). Hepatocytes are the primarysource of α₁PI, and in normal, healthy individuals, the range ofcirculating α₁PI is 20-53 μM between the 5^(th) and 95^(th) percentiles(Brandy et al., 1991; Bristow et al., 1998). However, during the acutephase of the inflammatory response, α₁PI may increase as much as 4-foldto 200 μM (Kushner, 1982). There are four common alleles of α₁PI, andthese are synthesized and secreted principally by hepatocytes (OMlM,2000). However, there are more than a hundred genetic variants, some ofwhich produce a molecule that prohibits secretion, and affectedindividuals manifest with 10-15% of the normal level of α₁PI in blood(Benninger, 1985). Individuals with this inherited form of α₁P1deficiency, especially males, are notably susceptible to respiratoryinfections and emphysema, and 80% who survive to adulthood succumb torespiratory failure between the fourth and sixth decades of life(Berninger, 1985). Prevalence is 0.03%, and α₁PI augmentation therapy inaffected individuals is the only approved therapeutic application ofα₁PI (OMIM, 2000).

Traditionally, α₁PI has been characterized as a proteinase inhibitorwhich has highest affinity for soluble granule-released elastase(HLE_(G)). Evidence now suggests α₁PI also interacts with cell surfaceHLE (HLE_(CS)) (Bristow et al., 2003; Tavor. S. et al., 2005). BothHLE_(CS) and HLE_(G) are synthesized and processed as a single molecularprotein; however, HLE is targeted exclusively for the cell surface earlyin ontogeny and for granule compartmentalization later in ontogeny(Gullberg et al., 1995; Garwicz et al., 2005). Mutations in the HLEencoding gene that result in decreased HLE expression produce periodiccycling in hematopoiesis that affect monocytes in the opposite phase toneutrophils (Horwitz et al., 1999; Horwitz et al., 2004). Mutations thatresult in increased HLE produce twice fewer absolute numbers ofcirculating CD4⁺ and CD8⁺ lymphocytes, and 7 times more monocytic cells(Person et al., 2003).

The proteinases and proteinase inhibitors that govern cell motility andhematopoiesis have evolved a different functional pattern in mice fromman, but there are many parallels. For example, in mice, it has beenshown that high concentrations of HLE accumulate in bone marrowfollowing granulocyte colony-stimulating factor (G-CSF) induced stemcell mobilization (Winkler et al., 2005). This accumulation was found toresult from the down-regulation of α₁PI expression. In man, the liver isthe primary source of both α₁PI and stem cells. As opposed to itsfunction to inhibit the enzymatic activity of HLE_(G), α₁PI binding toHLE_(CS) induces cell migration in a manner that does not appear toinvolve enzymatic activity (Wolf et al., 2003). The effect of α₁PI oncell motility is especially profound during migration of stem cells andearly progenitor cells. Hematopoiesis begins with stem cell migrationfrom fetal liver through the periphery to the stromal area ofhematopoietic tissue, retention, differentiation, and release ofmaturing progenitor cells back into the periphery. Migration of stemcells to, and myeloid-committed progenitor cells from bone marrow iscontrolled by HLE_(CS), the chemokine stromal cell-derived factor-1(SDF-1), and the SDF-1 receptor CXCR4 (Tavor. S. et al., 2005; Lapidotand Petit, 2002). Cell migration is dependent on the localization ofHLE_(CS) into podia formation at the leading edge of the cell (Tavor. S.et al., 2005; Cepinskas et al., 1999), and podia formation is induced bybinding of active α₁PI to HLE_(CS) in a manner that includesco-localization of HLE_(CS) with CD4 and CXCR4 (Bristow et al., 2003).The current method for therapeutic mobilization of myeloid-committedprogenitor cells from bone marrow is by the action of G-CSF, and it hasbeen shown that G-CSF mediates this activity by antagonizing CXCR4 andHLE_(CS) (Lapidot and Petit, 2002). The molecular mechanisms thatmobilize lymphoid-committed progenitors from hematopoietic tissue arenot known. Evidence described in this application now suggests activeα₁PI mediates this activity (Examples 1-3 below). Following treatmentwith α₁PI in animal models, the migration of transplanted human leukemiacells into circulation is decreased, but the migration of stem cells tohematopoietic tissue is increased (Tavor. S. et al., 2005). Theseresults suggest that α₁PI influences the migration of cells into and outof circulation depending, in part, on the stage of differentiation ofthe cell.

When bone marrow-derived erythroid progenitors cells (burst-formingunits-erythroid) are incubated with α₁PI in vitro, growth of immaturecells is significantly suppressed (42.5%±5.5%) (Graziadei et al., 1994).In contrast, growth of mature cells is unaffected by α₁PI (3.6%±3.4%).These results demonstrate that in addition to myeloid- andlymphoid-committed progenitors, α₁PI influences the genesis oferythroid-committed progenitor cells dependent on their stage ofdifferentiation.

Previous therapeutic application of α₁PI has been restricted toaugmentation in patients diagnosed with inherited α₁PI deficiency forthe purpose of ameliorating respiratory distress such as occurs inemphysema and chronic obstructive pulmonary disease (COPD). Considerableinterest in producing recombinant α₁PI has resulted in development ofseveral successful expression systems including bacterial and plant cellexpression as well as viral vector and oral delivery (Chowanadisai etal., 2003; Luisetti and Travis, 1996). Recombinant α₁PI is in phase Iclinical trials for augmentation in individuals with inherited α₁PIdeficiency (Flotte et al., 2004), and is in phase II clinical trials fortreatment of atopic dermatitis. Recombinant α₁PI has been tested forpreventing the onset of type I diabetes in genetically predisposed mice(Song et al., 2004). Nevertheless, there is a need in the art fordeveloping recombinant α₁PI with due consideration of itsconformation-dependent function to mobilize either lymphoid-lineage ormyeloid-lineage maturing cells. As recognized by the inventor herein,because α₁PI induces cell motility depending on its active orproteolytic ally modified conformation, various active and modifiedα₁PI's provide powerful new therapeutics for mobilizing targeted cellsubsets through tissue.

SUMMARY OF THE INVENTION

This invention is directed to the use of α₁PI and modified α₁PI tocontrol the phenotypic composition of circulating and tissue-associatedcells derived from hematopoietic stem cells.

Various modified α₁PI's are also provided. Screening methods andtreatment for abnormalities in the phenotypic profile of blood cells arealso provided. Such abnormalities are associated with, e.g., HIV-1infection, microbial infection, leukemia, solid tumor cancers,atherosclerosis, autoimmunity, stem cell transplantation, organtransplantation, and other diseases affected by cells of the immunesystem. The invention is based, in part, on a previously unrecognizedfundamental property of α₁PI to regulate the phenotypic composition ofcirculating and tissue-associated cells derived from hematopoietic stemcells.

Accordingly, this invention provides a method for identifying a modifiedα₁PI as suitable for use in treating a disease, disorder or condition ina subject, comprising: (a) producing the modified α₁PI; and (b)measuring a biological activity of the modified α₁PI in a biologicalassay for predicting effectiveness in treating the disease, disorder orcondition in the subject, wherein the modified α₁PI is identified assuitable for treating the disease, disorder or condition from a changein the biological activity relative to a control activity measured for awild-type α₁PI. In one embodiment, the modified α₁PI is produced bysite-directed mutagenesis, proteolysis, or both. In another embodiment,the disease, disorder or condition is selected from the group consistingof HIV-1 infection, bacterial infection, leukemia, a solid tumor,atherosclerosis, an autoimmune disease, organ transplantation, and stemcell transplantation. In another embodiment, the stem celltransplantation is autologous stem cell transplantation. In anotherembodiment, the biological assay is selected from the group consistingof an elastase inhibition assay, a receptor co-capping assay, a cellmotility assay, a lymphoid-committed progenitor cell mobilization assay,an HIV-1 gp120 antibody cross-reactivity assay, and an HIV-1 infectivityfacilitation assay. In another embodiment, the subject is a human or anon-human animal. In another embodiment, proteolysis comprisescontacting a wild-type or a recombinant α₁PI with a protease selectedfrom the group consisting of elastase, stromelysin-3, matrixmetalloproteinase, collagenase, gelatinase, pepsin, plasmin, urokinase,chymotrypsin, thrombin, CD26, complement component C1, and complementcomponent C3.

In another embodiment, site-directed mutagenesis comprises changing atleast two wild-type amino acid residues selected from the groupconsisting of residues 370-374 and 385 to a non-wild-type residue,wherein one changed residue is at position 385. In another embodiment,at least one amino acid selected from the group consisting of residues370-374 and 385 is changed from wild-type to glycine, threonine, or ahydrophobic amino acid. In another embodiment, the hydrophobic aminoacid is selected from the group consisting of isoleucine, leucine,phenylalanine, tyrosine and valine.

This invention provides a modified human α₁PI comprising a change in awild-type amino acid residue selected from the group consisting ofresidues 370-374 and 385. In one embodiment, the genetically modifiedα₁PI further comprises modification by proteolysis. In anotherembodiment, the wild-type amino acid residue is changed to glycine,threonine, or a hydrophobic amino acid. In another embodiment, thehydrophobic amino acid is selected from the group consisting ofisoleucine, leucine, phenylalanine, tyrosine and valine. In anotherembodiment, the modified human α₁PI comprises at least two changes inwild-type amino acid residues comprising a change at position 385 and achange at a position selected from the group consisting of positions370-374. In another embodiment, the methionine at position 385 ischanged to a non-methionine amino acid. In another embodiment, thenon-methionine amino acid is selected from the group consisting ofglycine, isoleucine, leucine, phenylalanine, threonine, and valine. Inanother embodiment, the modified human α₁PI is capable of a reducedbinding activity in an HIV-1 gp120 antibody cross-reactivity assay,relative to a wild-type α₁PI. In another embodiment, the residue changesin the modified human α₁PI comprise the following three amino acidsubstitutions: Phe372Gly; Leu373Gly; and Met 385Val. In anotherembodiment, the residue changes in the modified human α₁PI consist ofthe following three amino acid substitutions: Phe372Gly; Leu373Gly; andMet 385Val.

This invention provides a method of treating a disease, disorder orcondition in a subject in need of said treatment, comprisingadministering an effective amount of an unmodified or modified α₁PI tothe subject. In one embodiment, the modified α₁PI is produced bysite-directed mutagenesis, proteolysis, or both. In another embodiment,the disease, disorder or condition is selected from the group consistingof HIV-1 infection, bacterial infection, leukemia, a solid tumor,atherosclerosis, an autoimmune disease, organ transplantation, and stemcell transplantation. In another embodiment, the subject is a human or anon-human animal. In another embodiment, the modified α₁PI comprises achange in a wild-type amino acid residue selected from the groupconsisting of residues 370-374, and further comprises a change inmethionine at position 385. In another embodiment, methionine atposition 385 is changed to a non-methionine amino acid selected from thegroup consisting of glycine, isoleucine, leucine, phenylalanine,threonine, and valine. In another embodiment, the modified α₁PI iscapable of a reduced binding activity in an HIV-1 gp120 antibodycross-reactivity assay, relative to a wild-type α₁PI. In anotherembodiment, the amino acid changes in the modified α₁PI comprise thefollowing three amino acid substitutions: Phe372Gly; Leu373Gly; and Met385Val. In another embodiment, the amino acid changes in the modifiedα₁PI consist of the following three amino acid substitutions: Phe372Gly;Leu373Gly; and Met 385Val. In another embodiment, the treatment methodfurther comprises administration of HIV-1 antiretroviral therapy. Inanother embodiment, the effective amount of modified α₁PI is a doseequivalent to about 42 mg/kg of active wild-type α₁PI.

This invention provides a method of treating a disease, disorder orcondition in a subject in need of said treatment, comprisingadministering an effective amount of an active α₁PI to the subject,wherein the disease, disorder or condition is selected from the groupconsisting of HIV-1 infection, bacterial infection, leukemia, a solidtumor, atherosclerosis, an autoimmune disease, organ transplantation,and stem cell transplantation. In one embodiment, the stem celltransplantation is autologous stem cell transplantation.

This invention provides a method of treating a disease, disorder orcondition in a subject in need of said treatment, comprisingadministering an effective amount of an active α₁PI to the subject,wherein the subject is characterized as having an abnormal orineffective number of lymphocytes, monocytes, or dendritic cells.

This invention provides a method of treating a disease, disorder orcondition in a subject in need of said treatment, comprisingadministering an effective amount of an inactive α₁PI to the subject,wherein the disease, disorder or condition is selected from the groupconsisting of bacterial infection, neutropenia and immunosuppression.

This invention provides a method of treating a disease, disorder orcondition in a subject in need of said treatment, comprisingadministering an effective amount of an inactive α₁PI to the subject,wherein the subject is characterized as having an abnormal orineffective number of granulocytes, monocytes, dendritic cells,eosinophils, or basophils. In one embodiment, the subject is a human ora non-human animal.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1( a-c). Correlation of CD4 lymphocyte levels with active α₁PI,HLE_(CS) and SDF-1 in healthy individuals. (a) Increased active α₁PI anddecreased HLE_(CS) ⁺ lymphocytes predict increased CD4⁺ lymphocytes inhealthy subjects specifically selected to represent a wide spectrum ofα₁PI concentrations. CD4⁺ lymphocytes (%)=50.48+0.27*active α₁PI(μM)−2.67* HLE_(CS) ⁺ lymphocytes (%) (r²=0.937, p<0.05, n=6). (b)Increased active α₁PI and decreased HLE_(CS) ⁺ lymphocytes predictincreased CD4⁺ lymphocytes in healthy subjects representing the generalpopulation. CD4⁺ lymphocytes (%)=37.80+0.43*active α₁PI(μM)−1.56*HLE_(CS) ⁺ lymphocytes (%) (r²=0.803, p<0.05, n=16). WhenSDF-1 is included in the model, CD4⁺ lymphocytes (%)=44.46+0.54*activeα₁PI (μM)−1.65* HLE_(CS) ⁺ lymphocytes (%)−0.03* SDF-1 (μM) (r²=0.875,p<0.05, n=16). (c) Active α₁PI (▪) and CD4⁺ lymphocytes () increaseproportionally during the acute phase of an enteric infection in avolunteer who was otherwise healthy.

FIG. 2( a-e). Binding of anti-gp120 antibody to human, but notchimpanzee α₁PI. (a) Monoclonal antibody 3F5 binding to α₁PI in serafrom 18 healthy humans and 20 healthy chimpanzees was measured in ELISA.Antibody bound (A_(490 nm)) was normalized for the serum α₁PIconcentration in each specimen and is represented as A₄₉₀nm/α₁PI(μM).Binding of 3F5 was 8- to14-fold greater to human than to chimpanzee α₁PI(p<0.001). Measurements were repeated 6 times using 3F5 and once usingmonoclonal antibody 1C1. Representative measurements are depicted. Barsrepresent mean values. (b) The presence of IgG-α₁PI immune complexes insera (A_(490 nm)) was detected in 11 of 38 HIV-1 infected patients, butnot in sera from 9 healthy individuals, 20 healthy chimpanzees, nor in 2chimpanzees 42 months following HIV-1 inoculation. Serum collected fromhealthy volunteers into tubes containing clot activating additive wereexcluded from immune complex analysis because of buffer incompatibility.Measurements were repeated at least 3 times, and representative data aredepicted. Bars represent mean values. (c) Active α₁PI concentration inHIV-1 infected patients (median 17 μM) was significantly below normal(median 26 μM, p<0.001). Active α₁PI in sera from 20 healthy chimpanzees(median 35 μM) and 2 chimpanzees post-HIV-1 inoculation median (39 μM)was significantly greater (p<0.02) than from 18 human sera (median 26μM). Active α₁PI was measured in 8 serial dilutions of each serumsample. (d) Inactive α₁PI concentration in HIV-1 infected patients(median 19 μM) was above normal (median 4 μM, p<0.001). (e) Afterincubating sera from 5 healthy individuals with monoclonal antibody 3F5,active α₁PI (12±7 μM) was significantly lower than in control seraincubated with medium alone (18±7 μM, p<0.001). Bars represent meanvalues.

FIG. 3( a-d). Corresponding conformation at the 3F5-recognized domainsin α₁PI and CD4-complexed HIV-1 gp120. Structures for human α₁PI (1HP7)and CD4-complexed HW-1 gp120 (1RZJ) from the NCBI Molecular ModelingDatabase (MMDB) were analyzed using Cn3D software(www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). Small carbohydratestructures were already associated with 1RZJ in MMDB, and the threeassociated with 1HP7 were added using Adobe Photoshop. HIV-1 gp120 isdepicted from two perspectives (a,b) with two α-helices highlighted (aa21-39 and 306-313). The gp120 peptide immunogen used to raise 1C1 and3F5 (aa 300-321) is located at the C-terminus of gp120, and the linearsegment YKVV (aa 315-318) along with the M-17 and theoligosaccharide-linked NGT (aa 92-94), are within 8A° of theconformational epitope. The gp120-homologous domain in α₁PI is alsolocated at the C-terminus of the protein, and is depicted from twoperspectives (c,d) with the highlighted antiparallel β-sheet strand atthe base of the cleft (aa 369-389), as well as the α-helices that formthe mouth of the cleft (aa 28-44 and 259-277). M-385, whichdistinguishes human from chimpanzee α₁PI, is indicated along with GKVV(aa 386-389), the oligosaccharide, and oligosaccharide-linked NST (aa46-48). The proteinase reactive site M-358, is indicated fororientation.

FIG. 4. Correlation between CD4⁺ lymphocytes and active α₁PI levels inHIV-1 infected patients. In 23 patients with <500 HIV RNA copies/ml,CD4⁺ lymphocyte levels correlate with active α₁PI. Three parametersigmoid regression yields CD4(cells/μl)=1+e^(−(active α1PI(μM)−25)/11)), r²=0.927, n=23). CD4⁺lymphocyte levels also correlate with inactive α₁PI. Two parameterexponential decay regression yields CD4(cells/μl)=834*e^(−0.034 inactive α1PI(μM)), r²=0.906, n=23). Patientsreceiving protease inhibitor therapy are depicted by squares. All otherpatients are depicted by circles. In 13 patients with >500 HIV RNAcopies/ml, no correlation was found to exist between CD4⁺ lymphocytelevels and active α₁PI.

DETAILED DESCRIPTION OF THE INVENTION Definitions:

Human α₁PI—Alphα₁-Proteinase Inhibitor (Human) is a sterile, stable,lyophilized preparation of highly purified human alpha₁-proteinaseinhibitor (α₁PI) also known as alphα₁-antitrypsin derived from humanplasma. There are three products of alphα_(s)-Proteinase Inhibitor(Human) that are currently FDA approved for treatment. Prolastin®(www.prolastin.com) produced by Talecris Biotherapeutics(www.talecris.com), Zemaira® (www.zemaira.com) produced by ZLB Behring(www.zlbbehring.com), and Aralast™ (www.aralast.com) produced by BaxterHealthcare Corp.

Active α₁PI—the fraction of α₁PI in plasma or other fluids that has thecapacity to inhibit elastase activity.

Inactive α₁PI—the fraction of α₁PI in plasma or other fluids that doesnot have the capacity to inhibit elastase activity. Active α₁PI may beinactivated by proteolytic cleavage, proteinase complexing, antibodycomplexing, or oxidation.

Genetically modified α₁PI—active α₁PI synthesized from the cDNA encodinghuman α₁PI which has been modified by site-directed mutagenesis. Thereare no current recombinant products that have been FDA approved fortreatment.

Proteolytically modified α₁PI—active or genetically modified human α₁PIwhich has been further modified by limited proteolysis to generatefragments. Proteolytic modification inactivates α₁PI.

Pharmaceutical Composition—When formulated in a pharmaceuticalcomposition, the therapeutic compound of the invention can be admixedwith a pharmaceutically acceptable carrier or excipient. The phrase“pharmaceutically acceptable” refers to molecular entities andcompositions that are “generally regarded as safe”, e.g., that arephysiologically tolerable and do not typically produce an allergic orsimilar untoward reaction, such as gastric upset, dizziness and thelike, when administered to a human. Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a statement government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and, moreparticularly, in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the compound is administered.Such pharmaceutical carriers can be sterile liquids, such as water andoils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike. Water or aqueous solution saline solutions and aqueous dextroseand glycerol solutions are preferably employed as carriers, particularlyfor injectable solutions. Alternatively, the carrier can be a soliddosage form carrier, including but not limited to one or more of abinder (for compressed pills), a glidant, an encapsulating agent, aflavorant, and a colorant. Suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin.

1. Treatment population: Active α₁PI promotes migration of lymphocytesand monocytic cells expressing HLE_(CS) (Bristow et al., 2003) (Examples1-3 below). Inactive α₁PI promotes migration of neutrophils and cellsexpressing the LDL-receptor related protein, LRP (Kounnas et al., 1996;Weaver et al., 1997), Treatment with active human α₁PI is indicated inindividuals manifesting abnormal numbers of functional lymphocytes,monocytic cells, or dendritic cells such as in HIV-1 disease, stem celltransplantation, solid organ transplantation, autoimmune exacerbations,diabetes, leukemia, lymphoma, solid tumors, and, atherosclerosis.Treatment with inactive human α₁PI is indicated in individualsmanifesting abnormal numbers of functional granulocytic, monocyticcells, dendritic, eosinophilic, or basophilic cells such as in microbialinfection, neutropenia, and immunosuppressed patients. Treatment outcomeis determined as described below in Section 7 of the DetailedDescription.

2. Treatment regimen: According to the Prolastin® Product Monograph(www.prolastin.com/pdf/prolastin monograph.pdf), the Zemaira®prescribing information literature, and the Aralast™ prescribinginformation, the recommended dosage for α₁PI is repeated weeklyinfusions of 60 mg/kg at a rate of 0.08 ml/kg/minute leading to thehistorical target threshold of 11 μM α₁PI in serum. The ideal bloodthreshold is 34 μM α₁PI, but this level has not been achievedtherapeutically. Delivery is traditionally by infusion, but recombinantα₁PI is also produced for ingestion (Chowanadisai et al., 2003).

The specific activity of Zemaira® is 70%, Prolastin® is 35%, andAralast™ is 55% where specific activity is defined as inhibition ofporcine pancreatic elastase (PPE) as described in the package insert.Thus, the recommended dose of Zemaira® α₁PI may be stated as 42 mg/kgactive α₁PI, Prolastin® as 21 mg/kg, and Aralast™ as 33 mg/kg activeα₁PI. Conversely, the inactive fraction of Zemaira® is 30% or 18 mg/kg,of Prolastin® is 65% or 39 mg/kg, and of Aralast is 45% or 27 mg/kg

The dosage of genetically modified α₁PI is determined by its capacity toinhibit PPE as described in Section 6 of the Detailed Description (seealso, U.S. Pat. No. 6,887,678). In accordance with the recommendedtreatment regimen using wild-type α₁PI, subjects are infused withgenetically modified α₁PI at a dosage that is in the range of 1 to 420mg/kg active α₁PI, with a target blood threshold of 35 μM geneticallymodified α₁PI. In some cases, either active or genetically modified α₁PIare further modified by limited proteolytic cleavage to generatefragments that are chemotactic for myeloid-lineage cells. For example,in microbial infections that attend neutropenia, proteolyticallymodified α₁PI is used to recruit neutrophils into infected tissue. Inthis case, individuals are infused with proteolytically modified α₁PI atthe concentration that is equivalent to 39 mg/kg inactive α₁PI. Inaddition to being monitored for PPE inhibitory activity, proteolyticallymodified α₁PI is screened as described in Section 3.2 of the DetailedDescription for its capacity of to induce receptor capping and cellmotility of myeloid-lineage blood cells such as neutrophils.

3. Recombinant α₁PI: In addition to plasma-derived α₁PI, recombinantα₁PI has the capacity to mobilize progenitor cells. A bioengineered formof α₁PI has been shown to partition into two cleavage fragments (Jean etal., 1998), and an α₁PI-insulin-like growth factor chimera has beenshown to induce chemotaxis (Sandoval et al., 2003). Modifications ofrecombinant α₁PI provide an improvement specific to HIV-1 and otherdiseases for mobilization of specific progenitor cells.

3.1 Structural features of α₁PI: The following represents the fulllength amino acid sequence for α₁PI (accession # K01396) including the24 aa signal peptide:

−24 MPSSVSWGIL LLAGLCCLVP VSLA 1EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQS N STNI 51FFSPVSIATA FAMLSLGTKA DTHDEILEGL NF N LTEIPEA QIHEGFQELL 101RTLNQPDSQL QLTTGNGLFL SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT 151EEAKKQINDY VEKGTQGKIV DLVKELDRDT VFALVNYIFF KGKWERPFEV 201KDTEEEDFHV DQVTTVKVPM MKRLGMFNIQ HCKKLSSWVL LMKYLG N ATA 251IFFLPDEGKL QHLENELTHD IITKFLENED RRSASLHLPK LSITGTYDLK 301SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA 351MFLEAIPMSI PPEVKFNKPF VFLMIEQNTK SPLFMGKVVN PTQK

The known Asn-linked carboxylation sites (denoted in bold underlinedletters) are found at aa 46, 83, and 247 (Nu kiwa et al., 1986; Jeppssonet al., 1985). The oligosaccharide structure at each site is eithertri-antenary or bi-antenary, and the various combinations give theprotein a characteristic electrophoretic charge denoted as phenotypicsubtypes of the four common genotypic alleles, M1A, M1V, M2, and M3.

The frequencies in, US Caucasians of M1A, M1V, M2, and M3 are 0.20-0.23,0.44-0.49, 0.1-0.11, and 0.14-0.19, respectively, accounting for 95% ofthis population (Jeppsson et al., 1985). M1A is thought to be the oldestvariant, and M1V has a single aa substitution, at position 213, Ala toVal. The M3 allele has a single aa difference with M1V, Glu to Asp atposition 376. The M2 allele has a single aa difference with M3, Arg toHis at position 101.

More than a hundred genotypic alleles have been identified, but exceptfor the S and Z alleles, most of them are exceedingly rare (OMIM, 2000).The S allele, frequency 0.02-0.04, has a single aa substitution atposition 264, Glu to Val, and individuals homozygous for this allelemanifest 60% normal α₁PI blood levels, but are not at risk for emphysemaor other known diseases except in combination with the Z allele (Brantlyet al., 1991; Sifers et al., 1988). The Z allele, frequency 0.01-0.02,has a single aa substitution at position 342, Glu to Lys, andindividuals homozygous for this allele manifest 10% normal α₁PI bloodlevels, and are at risk for emphysema and autoimmunity.

3.2 Functional properties of α₁PI: There are three distinct activitiesof α₁PI that are determined by sites in the C-terminal region of α₁PIdefined herein as aa 357-394,

PMSI PPEVKFNKPF VFLMTIEQNTK SPLFMGKVVN PTQK.

The crystal structure for active α₁PI (1HP7, NIH NCBI Molecular ModelingDataBase mmdbId:15959) is depicted in FIG. 3 with Met (aa 358) and Met(aa 385) designated. The β-sheet formation of the C-terminal region ofα₁PI (aa 369-394) is designated. Two α-helix domains (aa 27-44 and257-280) shield the β-sheet domain in a manner resembling theantigen-binding cleft of the major histocompatibility complex.

The first activity of α₁PI is its well characterized proteinaseinhibition which is a property only of active, uncleaved α₁PI. Thereactive site for this activity is Met (aa 358) contained in the domainPro-Met-Ser-Ile-Pro (PMSIP, aa 357-361). Active α₁PI may be inactivatedby proteinase complexing, cleavage, or oxidation of Met (aa 358).Interaction at the scissile bond Met-Ser (aa 358-359) may be mediated bymany proteinases including HLE_(G). The two cleavage products of α₁PImay dissociate under some circumstances, but may remain associated in anew, rearranged configuration that may irreversibly incorporate HLE_(G),but may not incorporate other proteinases, for examplemetalloproteinases (Perkins et al., 1992).

The tertiary structure for the rearranged α₁PI configuration has notbeen solved (Mellet et al., 1998); however, X-ray diffraction andkinetic analyses of cleaved α₁PI suggest that the strand SIPPEVKFNKP (aa359-369) may separate 70A° from its original position and insert intothe β-sheet formation on the opposite face of the molecule (β-sheet A)in a manner that would significantly alter proteinase and receptorrecognition (Elliott et al., 2000). Thus, four configurations of theC-terminal region of α₁PI are thought to occur (Table 1).

TABLE 1 Functions of the C-terminal region of α₁PI Lymphoid anti- HIV-1Proteinase cell Myeloid cell gp120 entry co- Inhibition migrationmigration binding factor Native configuration in the active α₁PI + +− + + Rearranged configuration in cleaved − − Unknown + − α₁PI Complexedwith HLE in cleaved α₁PI − − + Unknown − Independent of other α₁PIcleavage − Unknown + Unknown Unknown products

Because the cleaved configuration of α₁PI lacks proteinase inhibitoryactivity, in deficient concentrations of active α₁PI, the result isemphysema and respiratory-related infections which are facilitated bythe presence of certain environmental factors, cigarette smoke,microbial factors, and inherited mutations that prohibit successfulproduction of active α₁PI.

A second activity of α₁PI is the stimulation of cell migration, and thisactivity is a property of both cleaved and uncleaved α₁PI. Cleaved α₁PIis recognized by LRP, and stimulates migration of myeloid-lineage cellsincluding neutrophils and monocytic cells (Joslin et al., 1992). Active,uncleaved α₁PI is recognized by HLE_(CS) and stimulates migration oflymphoid-lineage cells and myeloid-committed progenitor cells (Bristowet al., 2003). Cell migration is initiated by α₁PI-induced co-capping ofreceptors such as HLE_(CS), CXCR4, and CD4 into podia formation(Cepinskas et al., 1999; Banda et al., 1988). In addition to theparticipation of podia formation during cell migration, thisconfiguration is also the preferred binding site for HIV-1 (Bristow etal., 2003). The reactive site in α₁PI for this activity isPhe-Val-Phe-Leu-Met (FVFLM, aa 370-374).

A third non-physiologic activity of α₁PI is binding to antibodiesreactive with HIV-1 envelope protein gp120, and this activity results ininactivation of α₁PI and blocking of the other two activities describedabove. The anti-gp120 monoclonal antibodies 1C1 (Repligen, Inc.,Cambridge, Mass.) and 3F5 (hybridoma culture supernatant,0085-P3F5-D5-F8, Dr. Larry Arthur, NCI-Frederick) were previously shownto be reactive with an epitope near the gp120 C5 domain (Moore et al.,1994). The antibody cross-reactive site of human α₁PI is contained inthe domainPhe-Leu-Met-lle-Glu-Gln-Asn-Thr-Lys-Ser-Pro-Leu-Phe-Met-Gly-Lys-Val-Val(FLMIEQNTKSPLFMGKVV, aa 372-389) (Bristow et al., 2001) Chimpanzee α₁PI,which differs from human α₁PI by a single amino acid, Val (aa 385), doesnot bind anti-gp120, consistent with the ability of chimpanzees toresolve HIV-1 infection and regain normal CD4⁺ lymphocyte levels. Thissuggests that the anti-gp120 cross-reactive site in human α₁PI isdetermined primarily by the Met residue (aa 385).

3.3 Expression of recombinant α₁PI: Any method known in the art may beused for producing genetically modified α₁PI's according to theinvention. Two preferred methods are briefly described below forproducing such recombinant α₁PI's; one method allows expression of α₁PIin rice cells and the other allows bacterial expression. The cDNAencoding human α₁PI is obtained from a human cDNA bank by andamplification of the fragment in accession number K01396 using two PCRprimers: N-terminal primer 5′ GAGGATCCCCAGGGAGATGCTGCCCAGAA 3′ andC-terminal primer 5′CGCGCTCGAGYTATTTTTTGGGTGGGATTCACCAC 3′ as previouslydescribed (Courtney et al., 1984; Terashima et al., 1999; Jean et al.,1998).

For expression in rice cells, expression cassettes are prepared by usinga 1.1 kb NheI-PstI fragment, derived from p1AS1.5, is cloned into thevector pGEM5zf- (Promega, Madison, Wis.): ApaI, AatII, SphI, NcoI,SstII, EcoRV, SpeI, NotI, PstI, SalI, NdeI, Sad, MluI, NsiI at the SpeIand PstI sites to form pGEM5zf-(3D/NheI-PstI). The GEM5zf-(3D/NheI-PstI)is digested with PstI and Sad and ligated in two nonkinased 30mers withthe complementary sequences 5′ GCTTG ACCTG TAACT CGGGC CAGGC GAGCT 3′and 5′ CGCCT AGCCC GAGTT ACAGG TCAAG CAGCT 3′ to form p3DProSig. A 5-kbBamHI-KpnI fragment from lambda clone λOSg1 A is used as a terminator.Hygromycin resistance is obtained from the 3-kb BamHI fragmentcontaining the 35S promoter-Hph-NOS of the plasmid pMON410.

Microprojectile bombardment is applied for transforming a Japonica ricevariety TP309. The bombarded calli are then transferred to NB mediumcontaining 50 mg/l hygromycin and incubated in the dark at 25° C. for10±14 days. Rice cells are cultured at 28° C. (dark) using a shaker withrotation speed 115 rpm in the AA(+sucrose) media. The medium is changedevery 5 days to maintain cell lines. AA(-sucrose) is used for α₁PIexpression. A bioreactor is used for 2-1-scale culture. The reactor isoperated at 28° C. (dark) at agitation speed 30±50 rpm with aerationrate 100 ml/min. During the growth phase (10 days), the pH of the mediais controlled at pH 5.7, while in the production phase the PH is 5.7±6.3(un-controlled).

Recombinant α₁PI is purified using polyclonal anti-human α₁PI antibody(Enzyme Research Laboratories, South Bend, Ind.) immobilized toCNBr-activated Sepharose 4B with a concentration of 1.5 mg/ml gel. Thegel (3.5 ml) is packed in a column (inner diameter 1.26 cm), andequilibrated with 50 mM Tris-HCl buffer (pH 7.6). Crude medium isapplied to the column at 1.0 ml/min. Absorbance at 280nm is monitored atthe outlet of the column. After washing with the equilibrium buffer,α₁PI is eluted with 0.1N HCl solution. A peak fraction is collected, andits pH is immediately adjusted with 1M Tris-HCl buffer (pH 8.0). Thesemethods yield an estimated 5.7 mg α₁PI/g dry cell.

Alternatively, the α₁PI cDNA are expressed in Escherichia coli strainBL21 transformed with pDS56α₁PI/hf (Invitrogen, Carlsbad, Calif.).Protein expression is induced by addition of 1mM isopropylb-D-thiogalactoside, and cultures are grown overnight at 31° C. Thecells are washed in metal-chelation chromatography binding buffer (5 mMimidazole/0.5M NaCl/20 mM Tris, pH 7.9) and disrupted by cavitation. Theclarified and filtered supernatants containing soluble α₁PI variants areapplied to a Ni²⁺ tagarose column, and bound proteins are eluted with100 mM EDTA. The eluates are adjusted to 3.5M NaCl and applied to aphenyl-Sepharose column. The bound α₁PI/hf is eluted with 20 mMBis-Tris, pH 7.0 and concentrated (4 mg/ml fmal) by diafiltration in thesame buffer.

4. Genetic modification of active WTI: Recombinant active α1PI isexpressed according to the procedures described in Section 3 of theDetailed Description. Wild-type human α₁PI is modified genetically todiminish or enhance sequence-specific reactive sites. For example, inHIV-1 disease, therapeutic α1PI variants maintain its inhibition ofsoluble HLE_(G) and its induction of cell migration, but diminish itscapacity to facilitate HIV-1 entry and bind antibodies reactive withHIV-1.

The genetic modifications of interest are described in Section 4.1 ofthe Detailed Description. Site-directed mutagenesis of active α1PI isperformed using standard procedures which are well known in the art(e.g., Parfrey et al., 2003; Current Protocols in Molecular Biology,2002). For example, the DNA sequence encoding the human α1PI signalpeptide in pDS56α₁PI/hf is replaced with sequences encoding the epitope(FLAG)-tag by insertion of the annealed complimentary oligos 5′CTAGAGGATCCCATGGACTACAAGGACGACGATGACAAGGAA 3′ and5′GATCTTCCTTGTCATCGTCGTCCTTGTAGTCCATGGGATCCT 3′. The resulting cDNA issubcloned into pDS56-6His to generate pDS56α1PI/hf. To generatepDS56α1PI/hf carrying an amino acid substitution, the DNA sequencesencoding the wild-type amino acid are replaced by the complimentaryoligos coding for the amino acids described in Section 4.1 of theDetailed Description. The resulting ORFs directed cytosolic expressionof the recombinant proteins initiating with a Met followed by the Hisand FLAG tags and the mature sequences of mutant α1PI.

4.1 Genetic modification within the domain that determines cellmigration (FVFLM, aa 370-374) is prepared by site-directed mutagenesisof specific amino acids:

4.1.1 Phe (aa 370) to Ile, Leu, Val, Tyr, or Gly.

4.1.2 Val (aa 371) to Phe, Leu, Ile, or Gly.

4.1.3 Phe (aa 372) to Ile, Leu, Val, Tyr or Gly.

4.1.4 Leu (aa 373) to Ile, Val, Phe, or Gly.

4.1.5 Met (aa 374) to Phe, Thr, Ile, Leu, Val, or Gly.

4.2 Modification within the domain that determines HIV-1 gp120 antibodyrecognition is prepared by site-directed mutagenesis of Met (aa 385) toPhe, Thr, Be, Leu, Val, or Gly.

5. Proteolytic modification of active or recombinant α₁PI: Proteolyticfragments of chemotactic molecules, such as complement, thrombin, andα₁PI, impart a primordial system of immune clearance mediators producingdiscrete classes of cellular responses. The appearance of variantchemotactic molecules avails immediate recruitment ofpathogen-responsive immune cells as a direct function of the proteasesspecific to each pathogen. To replicate this system for therapeuticapplication, active or recombinant α₁PI are modified proteolytically todiminish or enhance conformation-specific reactive sites.

Cleavage of α₁PI producing inactive α₁PI maybe accomplished using avariety of proteinases. Cleavage by elastase is between Met-Ser (aa358-359) (Berninger, 1985), and by stromelysin-3, a stromal cell-derivedmatrix metalloproteinase (MMP), between Ala-Met (aa 350-351) (Pei etal., 1994). Cleavage of α₁PI by neutrophil collagenase or gelatinase isbetween Phe-Leu (aa 352-353) producing inactive α₁PI (Desrochers et al.,1992). Other MMPs have also been shown to cleave α₁PI (Mast et al.,1991). Significantly, α₁PI is cleaved by proteinase derived frompathogenic organisms such as Pseudomonas elastase (Barbey-Morel andPerlmutter, 1991).

The C-terminal α₁PI proteolytic fragments acquire attributes that allowinteraction with the LDL receptor-related protein (LRP) (Poller et al.,1995) and other receptors that recognize a pentapeptide sequence FVFLM(aa 370-374) (Joslin et al., 1992) in a manner that produces, chemotaxisof neutrophils, increased LDL binding to monocytes, upregulated LDLreceptors, increased cytokine production, and α₁PI synthesis (Banda etal., 1988; Janciauskiene et al., 1999; Janciauskiene et al., 1999). Ithas been shown that fibrillar aggregates of the C-terminal fragment ofα₁PI facilitate uptake of LDL by LRP on the hepatolastoma cell lineHepG2 (Janciauskiene and Lindgren, 1999), and these fragmentsparticipate in atherosclerosis (Dichtl et al., 2000). Specifically,active or recombinant α₁PI are incubated at the relevant optimalconditions with one or a combination of pepsin, plasmin, urokinase,chymotrypsin, thrombin, CD26, matrix metalloproteinases, complementcomponents C1 or C3, and other proteinases that facilitate thegeneration of chemotactic fragments of α₁PI (Methods in Enzymology,1970; Hooper, 2002). Cleavage of α₁PI is then terminated by changing theoptimal conditions in the proteinase mixture to conditions that preventproteinase activity, for example at temperature or pH extremes (Methodsin Enzymology, 1970; Hooper, 2002).

6. Functional capacity of active, genetically modified, andproteolytically modified α1PI: Various unmodified and modified α₁PI'sare screened and selected for use in treatment of specific diseases bydetermining their capacity in vitro and/or in vivo to perform thefollowing functions in the following assays:

6.1 Inhibit elastase: The procedures for measuring the capacity of α₁PIto inhibit soluble forms of porcine pancreatic elastase (PPE) or HLE_(G)are well established (U.S. Pat. No. 6,887,678) (Bristow et al., 1998).Briefly, PPE is incubated for 2 min with α1PI, and to this mixture isadded, the elastase substrate succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide(SA³NA). Results are detected by measuring the color change at 405nm.

In complex mixtures, α₁PI competes for binding to PPE with otherproteinase inhibitors or ligands present in the mixture. For example,PPE has higher affinity for α₂macroglobulin (α₂M) than for α₁PI, andwhen complexed with α₂M, PPE retains the ability to cleave smallsubstrates. In the presence of α₂M, PPE binds α₂M and is protected frominhibition by α₁PI, and the complexation of PPE with α₂M can be measuredby detecting the activity of PPE using

SA³NA. To measure the inhibitory capacity of α₁PI in complex mixturessuch as serum, two-fold serial dilutions of serum are incubated with aconstant, saturating concentration of PPE. The added PPE is bound by,α₂M and α₁PI in the diluted serum dependant on their concentrations, thegreater the concentration of serum, the greater the concentration of α₂Mand α₁PI. Since there is more α₁PI in serum than α₂M, as serum isdiluted, α₂M is diluted out, and in the absence of α₂M, PPE is bound andinhibited by α₁PI. The complexation of PPE with α₁PI can be measured bydetecting the loss of activity of PPE using SA³NA. As serum is furtherdiluted, α₁PI is also diluted out, and the loss of complexation of PPEwith α₁PI can be measured by detecting the gain in activity of PPE usingSA³NA. The plot of PPE activity versus serum dilution makes a V shapedcurve, PPE activity first decreasing as serum is diluted, and thenincreasing as serum is further diluted. The nadir of PPE activity isused to calculate the precise concentration of active α₁PI in themixture (Bristow et al., 1998).

6.2 Induce receptor co-capping and cell motility: The procedures forinducing receptor capping have been described (Bristow et al., 2003).The cells of interest (monocytes, lymphocytes, neutrophils, or otherblood cells, e.g. leukemic cells) are isolated from blood or tissueusing standard techniques (Messmer et al., 2002) and examined forreactivity with α₁PI.

To examine receptor capping, cells are incubated with active or modifiedα₁PI for 15 min in humidified 5% CO₂ at 37° C. Cells are applied to thesample chambers of a cytospin apparatus (Shandon Inc. Pittsburgh, Pa.),and slides are centrifuged at 850 rpm for 3 min. Slides are fixed byapplication of 50 μl 10% formalin to the sample chambers of the cytospinapparatus followed by an additional centrifugation at 850 rpm for 5 min.Slides are incubated for 90 min at 20° C. with fluorescently-labeledmonoclonal antibodies having specificity for the receptors of interestand examined by microscopy.

Cell motility results from selective and sequential adherence andrelease produced by activation and deactivation of receptors (Wright andMeyer, 1986; Ali et al., 1996), consequent polar segregation of relatedmembrane proteins to the leading edge or trailing uropod, and bothclockwise and counterclockwise propagation of Ca⁺⁺ waves which initiatefrom different locations in the cell (Kindzelskii and Petty, 2003).Thus, several aspects of the complex process may be quantitated. Themost direct and most easily interpreted method for quantitating cellmotility is the enumeration of adherent cells in response to achemotactic agent such as α₁PI.

For detecting adherence, sterile coverslips are washed in endotoxin-freewater, and to each coverslip is delivered various dilutions of active ormodified α₁PI. Cells are subsequently delivered to the coverslips, mixedto uniformity with α₁PI, and incubated for 30 min in humidified 5% CO₂at 37° C. without dehydration. After stringently washing the coverslipsfree of non-adherent cells, adherent cells are fixed by incubation for10 min at 20° C. with 4% paraformaldehyde containing 2.5 mM of thenuclear staining fluorescent dye, acridine orange(3,6-bis[dimethylamino]acridine. Slides are examined by microscopy, andmeans and standard deviations are determined by counting adherent cellsin at least three fields/coverslip.

6.3 Mobilize lymphoid-committed progenitor cells: In the nonobesediabetic/severe combined immunodeficiency (NOD/SCID) mouse model, bonemarrow-engrafted human cells can be mobilized by G-CSF (Petit et al.,2002). This model is adapted to assess the capacity of active ormodified α₁PI to mobilize human lymphoid- or myeloid-lineage cells,respectively.

NOD/SCID mice are housed under defined flora conditions in individuallyventilated (HEPA-filtered air) sterile micro-isolator cages. Humanchimeric mice are obtained after sublethal irradiation (375 cGy at 67cGy/min) and injection of 2×10⁷ human cord blood mononuclear cells. Fourto five weeks post transplantation, mobilization is performed byapplication of either G-CSF or α₁PI. For mobilization ofmyeloid-committed progenitors, mice receive daily subcutaneousinjections of 300 mg/kg G-CSF (Filgrastim, Neupogen® or Neulasta®,Amgen, Inc.) in 250 μl of 0.9% NaCl, 5% fetal calf serum for 4-5 days.Alternatively, mice receive twice weekly infusion via the dorsal tailvein of inactive or modified α₁PI (39 mg/kg) at a rate of 0.08ml/kg/minute. For mobilization of lymphoid-committed progenitors, micereceive twice weekly infusion via the dorsal tail vein of active ormodified α₁PI (42 mg/kg) at a rate of 0.08 ml/kg/minute. Mice areasphyxiated with dry ice, peripheral blood is collected by cardiacaspiration into heparinized tubes, and bone marrow is harvested, andcells are flushed from femurs and tibias into single-cell suspensions.Peripheral blood and bone marrow cells are analyzed by flow cytometryfor the presence of myeloid and lymphoid markers including CD34 CD38,CD10, CD11b, CD11c, CD13, CD14, CD19, CD3, CD4, CD8, CD45, CD184(CXCR4), CD66, and HLE_(CS) (U.S. Pat. No. 6,858,400).

6.4 Bind anti-HIV-1 gp120: Active or modified α₁PI are incubated influid phase with monoclonal antibodies reactive with HIV-1 gp120. Theanti-gp120 monoclonal antibodies 3F5 (hybridoma culture supernatant,0085-P3F5-D5-F8) is reactive with an epitope near the gp120 C5 domain(Moore et al., 1994). Clone α70 (ICN Biochemicals, Aurora, Ohio) isreactive with the V3-loop of gp120, a domain that is identical to theHLE ligand inter-a-trypsin inhibitor (Pratt et al., 1987). Immunecomplexes are captured by incubating mixtures in wells of a microtiterplate pre-coated with chicken anti-human α₁PI IgG. Binding is detectedusing horse radish peroxidase-conjugated rabbit anti-mouse IgG followedby substrate, orthophenylene diamine HCl.

6.5 Facilitate HIV-1 infectivity: Primary non-syncytium inducing HIV-1clinical isolates (Advanced Biotechnologies, Rivers Park, Ill.) are usedto infect peripheral blood mononuclear cells maintained in wells of a 96well tissue culture plate at 2×10⁶cells/ml in RPMI-1640 containing 20%autologous serum and 10% IL-2 (Cellular Products, Buffalo, N.Y.). Priorto addition of HIV-1, cells are incubated with active or modified α₁PIfor 0 min or 60 min at 37° C., 5% CO₂. In vitro infectivity outcome isdetermined in triplicate by p24 accumulation or by RT activity aspreviously described (Bristow, 2001). Cell counts and viability aredetermined at the final time point.

7. Treatment outcome measurements:

7.1 To determine the effectiveness of treatment on elastase inhibitorycapacity, individuals are monitored weekly for active and inactive α₁PIblood levels (Bristow et al., 1998) (U.S. Pat. No. 6,887,678). Briefly,a constant amount of active site-titrated PPE is allowed to incubatewith serial dilutions of serum for 2 min at 37° C. after which a PPEsubstrate is added. Determination of the molecules of substrate cleavedby residual, uninhibited PPE is used to calculate the molecules ofactive and inactive α₁PI in blood.

7.2 To determine the effectiveness of treatment on inducing changes inlevels of targeted blood cell populations, treated individuals aremonitored weekly for changes in complete blood count and differential,as well as for changes in specific subsets of blood cells such as CD4⁺cells and HLE_(CS) ⁺ cells using flow cytometry (Bristow et al., 2001;Bristow, 2001) (U.S. Pat. NO. 6,858,400). Briefly, 100 μl of whole bloodis incubated with a panel of fluorescently-labeled monoclonal antibodiesapproved by the FDA for medical diagnostics. These antibodies areselected to specifically recognize the cell receptors that uniquelyidentify the cell population of interest. Identification and enumerationof the cells in blood that are bound to the monoclonal antibodies isperformed using flow cytometry.

7.3 To determine the influence of treatment on disease progression,individuals are monitored for the specific pathologic determinants ofdisease which are well known in the art for the various indications,e.g. in stem cell transplantation, organ transplantation, autoimmunity,diabetes, leukemia, cancer, HIV-1 infection, atherosclerosis, and otherdiseases influenced by blood cells. For example, in HIV-1 disease,individuals are monitored for changes in CD4⁺ lymphocyte levels and HWlevels (Bristow et al., 2001; Bristow, 2001), In leukemia or cancer,individuals are monitored for changes in the presence of leukemic orcancerous cells (Tavor. S. et al., 2005). In stem cell transplantation,individuals are monitored for changes in normal blood cells (Jansen etal., 2005). In organ transplantation, individuals are monitored fororgan rejection (Kirschfink, 2002). In autoimmunity, individuals aremonitored for the presence of autoantibodies and specific functions ofthe affected organs (Marinaki et al., 2005). In diabetes andatherosclerosis, individuals are monitored for changes in totalcholesterol, LDL, HDL, and triglyceride levels (Talmud et al., 2003).

EXAMPLES

1. Increased CD4⁺ lymphocytes are correlated with increased α₁PI anddecreased HLE_(CS) ⁺ lymphocytes in healthy individuals. In healthyindividuals, circulating α₁PI ranges from 18-53 μM between the 5^(th)and 95^(th) percentiles, and 90-100% of this protein is in its activeform as determined by inhibition of porcine pancreatic elastase (Bristowet al., 2001). To investigate the relationship between active α₁PI,HLE_(CS) ⁺, and CD4⁺ lymphocytes, 6 healthy HIV-1 seronegative adults, 3males and 3 females, were specifically selected to represent a widespectrum of α₁PI (1.9-61.5 μM). Subjects were measured for CD4, CXCR4,CCR5, HLE_(CS), active and inactive α₁PI levels, and α₂-macroglobulin(α₂M). Independently, neither active α₁PI, HLE_(CS) ⁺ lymphocytes, α₂M,CXCR4⁺ lymphocytes, nor CCR5⁺ lymphocytes were correlated with CD4⁺lymphocytes. However, by multilinear regression analysis, it was foundthat higher numbers of CD4⁺ lymphocytes (% lymphocytes) were correlated(r²=0.937) with two counterbalancing variables together, higher activeα₁PI (p=0.008) and lower HLE_(CS) ⁺ lymphocytes (p=0.034) (FIG. 1 a).

To investigate CD4⁺ lymphocyte levels in the general population, bloodwas collected from an additional 18 healthy, HIV-1 seronegative adults,9 males and 9 females, who were measured for CD4, CXCR4, CCR5, SDF-1,active and inactive α₁PI levels. HLE_(CS) was measured in 16 of theseindividuals. Values for active α₁PI (19-37 μM) and SDF-1 levels(191-359pM) for these volunteers were found to be within normal ranges.Higher CD4⁺ lymphocytes (%) were again found to be correlated (r²=0.803)with higher active α₁PI (p<0.007) and lower HLE_(CS) ⁺ lymphocytes(p<0.001) (FIG. 1 b). Along with active α₁PI and HLE_(CS) ⁺ lymphocytes,lower SDF-1 concentration (p=0.02) also significantly contributed topredicting higher CD4⁺ lymphocytes (r²=0.875). Although CXCR4⁺lymphocytes were not significantly related to CD4⁺ lymphocytes, this mayreflect the detection of both active and inactive configurations ofCXCR4 on individual cells (Percherancier et al., 2005).

There was no statistical difference between the volunteers in FIG. 1 aand b in their active α₁PI levels (median=23 and 24, respectively) andCD4⁺ lymphocytes (mean=48% and 45%, respectively); however, involunteers depicted in FIG. 1 a the range is wide (1.9-61.5 μM) andstandard deviation is large (s=24),whereas in volunteers depicted inFIG. 1 b, the range of active α_(i)PI is narrow (19-37 μM) and thestandard deviation is small (s=6), and this suggests CD4⁺ lymphocytelevels are sensitive to small differences in α₁PI levels. Thesensitivity of CD4⁺ lymphocyte levels to α₁PI levels is furtherexemplified during the acute phase of an enteric infection in onevolunteer who was otherwise healthy (FIG. 1 c). In this individual, anincrease in total lymphocytes (1.15-fold) and CD4⁺ lymphocytes (1.2fold) was found to occur in concert with an increase in total α₁PI(2.7-fold), increase in active α₁PI (1.5 fold), and decrease in HLE_(CS)(2.9-fold).

2. Monoclonal anti-gp120 binds human, but not chimpanzee α₁PI. Twomonoclonal antibodies (1C1 and 3F5) which bind a conformationallydetermined epitope near the C5 domain of gp120 (Moore et al., 1994) werefound to also bind human α₁PI (Bristow et al., 2001). It washypothesized that anti-gp120 mediated depletion of active α₁PI might bepathognomonic for HI-1 AIDS. If true, chimpanzee α₁PI should differ fromhuman α₁PI since HIV-1 infected chimpanzees survive infection and regainnormal levels of CD4⁺ lymphocytes (Rutjens et al., 2003). Sequencecomparison revealed that human α₁PI differs from chimpanzee α₁PI by oneamino acid (aa 385) caused by a single nucleotide change (NCBI accessionnumbers BT019455 and XP_(—)522938), and this aa difference lies in thegp120-homologous region of α₁PI. To determine whether this sequencedifference affects the binding of anti-gp120 to α₁PI, 20 human and 20chimpanzee sera were compared. Both 1C1 (data not shown) and 3F5exhibited 8- to 14-fold greater binding to human, than chimpanzee α₁PIin 6 repeat measurements (p<0.001) (FIG. 2 a). Negative controlmonoclonal antibody α70 which reacts with the V3-loop of gp120 failed tobind human α₁PI (data not shown) consistent with previous findings(Bristow et al., 2001). Serum α₁PI in two human subjects exhibited muchgreater affinity for 3F5 than that from other subjects, and thissuggests the epitope of α₁PI recognized by 3F5 may be phenotypicallydetermined. When these two subjects were omitted from the comparison,the statistical difference between binding of 3F5 to human or chimpanzeeα₁PI was maintained (p<0.001).

To examine the relationship between lower CD4⁺ lymphocyte levels andlower active α₁PI levels in HIV-1 disease, blood from 38 HIV-1 infectedpatients was analyzed. Of these 38 patients, 29% had detectable IgG-α₁PIimmune complexes, 89% were on antiretroviral therapy, and 60% had <500HIV-1 RNA copies/ml (FIG. 2 b). The number of patients exhibitingdetectable IgG-α₁PI immune complexes in this study differs from aprevious study of 68 HIV-1 patients in which 60% had detectable IgG-α₁PIimmune complexes, 53% were on antiretroviral therapy (AZT only), and 16%had <500 HIV-1 RNA copies/ml (Bristow et al., 2001). This reason forthis difference may be related to the improved antiretroviral therapy inplace today.

None of the sera from healthy chimpanzees, nor sera collected from 2chimpanzees post-HIV-1 inoculation, had evidence of detectable IgG-α₁PIimmune complexes. The HIV-1 inoculated chimpanzees were confirmed to beHIV-1 infected, but had normal CD4⁺ lymphocytes (Girard et al., 1998).In addition, despite the presence of anti-gp120, we found no evidence ofIgG-α₁PI immune complexes in 10 rhesus macaques following immunizationwith simian/human immunodeficiency virus (SHIV 89.6) gp120 or gp140, orin 3 macaques infected with SHIV (data not shown). Extensive in vitroanalyses failed to demonstrate bi-molecular complexes between gp120 andα₁PI (data not shown), and the absence of detection of IgG-α₁PI immunecomplexes in sera from HIV-1 infected chimpanzees suggests gp120 andα₁PI are not associated by aggregation in sera. These results suggestthat IgG-α₁PI immune complexes are unique to HIV-1 disease in humans.

Consistent with evidence from a previous patient study, active α₁PI inthe HIV-1 infected patients was significantly below normal (median 17μM, p<0.001) (FIG. 2 c) and inactive α₁PI was significantly above normal(median 19 μM, p<0.001) (FIG. 2 d) (Bristow et al., 2001). In contrastto humans, active α₁PI levels in sera collected from the 2 chimpanzeespost-HIV-1 inoculation (39 μM) were not different from normal chimpanzeeand human sera (p=0.810) (FIG. 2 c).

To determine whether α₁PI becomes inactivated after complexing with the3F5 anti-gp120 monoclonal antibody, 3F5 was incubated with sera samplesfrom five healthy individuals. In comparison to untreated sera, α₁PIactivity was significantly diminished to the same degree in all sera(mean difference=5.8±0.5 μM, p<0.001) (FIG. 2 e).

The gp120 epitope recognized by 1C1 and 3F5 is considered to beconformation-dependent (Moore et al., 1994). The gp120 peptide immunogenused to raise 1C1 and 3F5 (aa 300-321, GGGDMRDNWRSELYKYKVVK) (Ratner etal., 1985) contains both an α-helix (aa 306-313) and linear strand (aa314-321) (FIG. 3 a,b), but other epitope determinants of the antibodiesare not known. In human α₁PI (FIG. 3 c,d), the gp120-homologous sequence(aa 369-389, PFVFLMIDQNTKSPLFMGKVV) folds to form a two-strandedantiparallel β-sheet that lies at the base of a cleft (4A° deep by 20A°long by 5A° wide) topped by two α-helices (aa 28-47 and 259-277) in asmaller, but similar configuration as the antigen-binding cleft of MHC(10A° deep by 25A° long by 10A° wide) (Bjorkman et al., 1987). At thefar end of the first of these α-helices is the N-linkedmannose-containing oligosaccharide that confers structural polymorphismto α₁PI (aa 46) (Jeppsson et al., 1985). In the center of the β-sheetthat lies in the cleft, is M-385 which distinguishes human fromchimpanzee α₁PI (V-385). The function of this cleft is not known, but asequence in the center of the β-sheet formation (aa 370-374, FVFLM) ishomologous to the fusion domain of HIV-1 gp41, and this sequence hasbeen implicated in binding HLE_(CS) (Bristow et al., 1995; Bristow etal., 2003) and stimulating cell motility (Joslin et al., 1991).

In α₁PI, the sequence GKVV (aa 386-389) lies within 5A° of M-385, N-46,and the N-linked oligosaccharide in a space occupying 5A° by 5A° by 5A°. In gp120, in the same relative orientation as in α₁PI, the sequenceYKVV (aa 315-318) lies within 5A° of M-17 and 8A° of N-92 and theN-linked mannose-containing oligosaccharide (Leonard et al., 1987) in aspace occupying 5A° by 5A° by 8A° . Evidence that α₁PI polymorphisms mayinfluence 3F5 binding (FIG. 2 a) suggests the polymorphism-determiningN-46 oligosaccharide participates in 3F5 recognition of α₁PI. Thus, the3F5 conformational epitope is suggested by these analyses to occupy a5A° by 5A° by 8A° space including KVV, M, N, and the N-linkedoligosaccharide. This proposed conformational epitope is consistent withpreviously characterized antigen configurations that containoligosaccharide determinants (Cygler et al., 1991) as well as withresults demonstrating that gp120 N-92 is invariant (Wei et al., 2003).

3. Active %PI is rate limiting for CD4+ lymphocytes in HIV-1 disease. Ofthe 36 patients included in the study population, 23 were below 500 and13 were above 500 HIV RNA copies/ml at the time of blood collection. Allpatients were measured for CD4, CXCR4, CCR5, SDF-1 levels, active andinactive α₁PI. Only 28 of these patients were additionally measured forHLE_(CS). Neither CXCR4 nor CCR5 were found to correlate individually orin combination with any parameters of disease being investigated inthese patients. Eleven of the 38 HIV-1 patients had active liver diseaseas defined by detectable Hepatitis B or C, or elevated liver enzymes.HIV-1 patients with liver disease were not different from patientswithout liver disease in active α₁PI (p=0.95), total α₁PI (p=0.79),CXCR4 (p=0.63), or CCR5 (p=0.9), but exhibited significantly higherSDF-1 (p<0.001), HLE_(CS) ⁺ lymphocytes (p<0.001), and CD4⁺ lymphocytes(p=0.04).

In the 23 patients with <500 HIV-1 RNA copies/ml, higher CD4⁺ lymphocytelevels were correlated with higher active α₁PI concentration (r²=0.927)and lower inactive α₁PI concentration (r²=0.946) (FIG. 4). Prediction ofCD4 levels from active α₁PI levels with 95% confidence had a standarderror of 151 cells/μl, and prediction from inactive α₁PI levels with 95%confidence had a standard error of 105 cells/4 Of these 23 patients,only 16 had been additionally measured for HLE_(CS). As in Healthyindividuals (FIG. 1), lower HLE_(CS) ⁺ lymphocytes was itself notcorrelated with higher CD4⁺ lymphocytes, but in combination with higheractive α₁PI was significantly correlated (p=0.01). That CD4⁺ lymphocytelevels could be predicted by active α₁PI alone with such a high degreeof accuracy in patients controlling their viral load suggests that,unlike the normal population, active α₁PI is rate limiting for CD4⁺lymphocyte levels in HIV-1 disease. In patients with >500 HIV RNAcopies/ml, there was no relationship between CD4⁺ lymphocyte levels andactive or inactive α₁PI (FIG. 4), and this suggests either HIV-1 itself,or other host processes had contributed to disrupting the regulation ofCD4⁺ lymphocyte levels.

4. α₁PI augmentation therapy in HIV-1 infected patients. The number ofCD4⁺ T lymphocytes in patients with <500 HIV-1 RNA copies/ml iscontrolled by their circulating concentration of α₁PI (Example 2). Thesepatients have below normal levels of circulating α₁PI (Bristow et al.,2001). Approximately 10% clinic patients in New York City who have_(<)500 HIV-1 RNA copies/ml also have <200 CD4 cells/μl, and thesepatients benefit from α₁PI augmentation by increasing their CD4⁺ Tlymphocyte numbers. Treatment of HIV-1 infected patients with α₁PIaugmentation is indicated in patients who are simultaneously receivingone or a combination of the four currently known classes, nucleosidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, HIV-1 aspartyl protease inhibitors, and fusion inhibitors.

Patients with <500 HIV-1 RNA copies/ml and <200 CD4 cells/μl who arereceiving antiretroviral therapy are treated using Zemaira® α₁PI.Patients receive weekly infusions of Zemaira® at 60 mg/kg as describedin Section 2 of the Detailed Description. Treatment outcome is monitoredas described in Section 7.3 of the Detailed Description. Specifically,patients receiving Zemaira® are monitored weekly for changes in activeand inactive α₁PI levels as well as for CD4⁺ T lymphocytes and othersubsets of circulating blood cells. Patients are also monitored forchanges in HIV-1 RNA copies/ml, LDL, HDL, cholesterol, triglycerides,and the occurrence of infections designated by the CDC as parameters ofHIV-1 disease progression (Castro et al., 1992). To determine possibleadverse effects of immune complex disease, individuals are monitored forthe presence of antibodies reactive with α₁PI as well as for theoccurrence of glomerulonephritis by measuring either proteinuria orserum creatinine levels (Bristow et al., 2001; Virella et al., 1981).

5. α₁PI augmentation therapy in HIV-1 infected patients usinggenetically modified α₁PI. Antibodies that recognize HIV-1 are the onlydiagnostic marker of infectivity. The presence of an anti-gp120 antibodythat also binds α₁PI has been detected in most HIV-1 infectedindividuals (Bristow et al., 2001), and this antibody inactivates andproduces deficient levels of α₁PI. Anti-gp120 does not bind chimpanzeeα₁PI which differs from human α₁PI by a single amino acid (aa 385)(Example 2). To therapeutically augment α₁PI in HIV-1 infectedindividuals, it is desirable to use genetically modified α₁PI whichsubstitutes a different aa in place of Met (aa 385). In addition, ahydrophobic domain (aa 370-374) near Met (aa 385) has been shown tofacilitate HIV-1 entry (Bristow et al., 2001). Thus, it is alsodesirable to change one or more of the aa in this hydrophobic domain fortreatment in HIV-1 disease.

α₁PI is genetically modified as described in Section 4 of the DetailedDescription with three substitutions, aa 385 (Met to Val), aa 372 (Pheto Gly), and aa 373 (Leu to Gly), and is designated α₁PI.β.F372G.L373G.M385V (α₁PI.β). The α₁PI.β sequence with aa changesrepresented in bold underlined letters is as follows:

−24 MPSSVSWGIL LLAGLCCLVP VSLA 1EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI 51FFSPVSIATA FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL 101RTLNQPDSQL QLTTGNGLFL SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT 151EEAKKQINDY VEKGTQGKIV DLVKELDRDT VFALVNYIFF KGKWERPFEV 201KDTEEEDFHV DQVTTVKVPM MKRLGMFNIQ HCKKLSSWVL LMKYLGNATA 251IFFLPDEGKL QHLENELTHD IITKFLENED RRSASLHLPK LSITGTYDLK 301SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA 351MFLEAIPMSI PPEVKFNKPF V GG MIEQNTK SPLF V GKVVN PTQK

The functional capacity of α₁PI.β depicted in Table 2 is determined asdescribed in Section 6 of the Detailed Description.

TABLE 2 Functions of the C-terminal region of α₁PI.β Lymphoid Proteinasecell Myeloid cell anti-gp120 HIV-1 entry Inhibition migration migrationbinding co-factor Native configuration in the active α₁PI.β + − − − +Rearranged configuration in cleaved α₁PI.β − − − − − Complexed with HLEin cleaved α₁PI.β − − − − − Independent of other α₁PI.β cleavage − − − −Unknown products

The recommended dose of α₁PI is 60 mg/kg. The specific activity ofZemaira® is 70%, where specific activity is defined as inhibition of PPE(Bristow et al., 1998). Thus, the recommended dose of Zemaira® α₁PI maybe stated as 42 mg/kg active α₁PI. In accordance with the recommendedZemaira® treatment regimen, HIV-1 patients with <500 HIV-1 RNA copies/mland <200 CD4 cells/μl who are receiving antiretroviral therapy areinfused with the concentration of α₁PI.β that is in the range of 1 to420 mg/kg active α₁PI with a target blood threshold of 35 μM α₁PI.β.Treatment of HIV-1 infected patients with α₁PI.β is indicated inpatients who are simultaneously receiving one or a combination of thefour currently known classes, nucleoside reverse transcriptaseinhibitors, non-nucleoside reverse transcriptase inhibitors, HIV-1aspartyl protease inhibitors, and fusion inhibitors. Treatment outcomeis monitored as described in Section 7.3 of the Detailed Description.Specifically, patients receiving α₁PI.β are monitored weekly for changesin active and inactive α₁PI levels as well as for CD4⁺ T lymphocytes andother subsets of circulating blood cells. Patients are also monitoredfor changes in HIV-1 RNA copies/nil, LDL, HDL, cholesterol,triglycerides, and the occurrence of infections designated by the CDC asparameters of HIV-1 disease progression (Castro et al., 1992). Todetermine possible adverse effects of immune complex disease,individuals are monitored for the presence of antibodies reactive withα₁PI as well as for the occurrence of glomerulonephritis by measuringeither proteinuria or serum creatinine levels (Bristow et al., 2001;Virella et al., 1981).

6. α₁PI inhibits SDF-1 induced migration of human leukemic cells in, butenhances migration of human stem cells. Human acute myeloid leukemiacells (AML) not only secrete HLE_(G), but also express HLE_(CS)constitutively on the cell surface in a manner that is regulated by theCXCR4/SDF-1 axis (Tavor. S. et al., 2005). Preincubation of AML cellswith α₁PI significantly reduced their SDF-1 dependent migration in allAML cells tested using an in vitro transwell assay (Tavor. S. et al.,2005). Further, in a mouse model it was found that α₁PI inhibited homingof transplanted human stem cells to bone marrow and egress oftransplanted AML cells from bone marrow. The influence of α₁PI was shownto occur by its action on HLE_(CS). When AML cells were treated withα₁PI, SDF-1 induced pseudopodia formation was prevented. These resultsare in contrast to previous studies using a U937 promonocytic cell linewhich demonstrated that α₁PI-induced pseudopodia formation was preventedby pretreatment with SDF-1 (Bristow et al., 2003), and this differenceemphasizes the importance of α₁PI and SDF-1 in promoting cell migrationof various cells dependent on their stage of differentiation.Augmentation with active and modified α₁PI is used therapeutically tocontrol the proliferation and spread of leukemia and lymphoma cells.Active α₁PI is used to prevent proliferation and spread of leukemia andlymphoma cells triggered by SDF-1. Inactive α₁PI is used to preventproliferation and spread of leukemia and lymphoma cells triggered byactive α₁PI. Patients receive therapeutic augmentation with active ormodified α₁PI with a target blood threshold of 35 μM active α₁PI and aremonitored for active and inactive α₁PI levels as well as for changes inthe number of AML cells in circulation using flow cytometry.

7. α₁PI augmentation therapy in patients with a microbial infection.High levels of neutrophils and HLE_(G) are present in the respiratorysecretions of patients with cystic fibrosis. The primary cause of thisinflammatory situation is chronic infection with Pseudomonas aeruginosaand other bacteria. Abundant α₁PI is present in these patients, but ispredominantly inactivated by HLE_(G) and P. aeruginosa elastase(Barbey-Morel and Perlmutter, 1991). Prolastin® has demonstratedimprovement by reducing elastase activity, neutrophil counts, andbacterial colonies in a rat model (Cantin and Woods, 1999). Inactivatedα₁PI is a chemo-attractant for neutrophils (Joslin et al., 1992). Inaddition to the therapeutic benefit of inhibiting the elevated elastaseactivity that attends the inflammatory sequelae of microbial infection,augmentation with active α₁PI diminishes the inactivated α₁PI-inducedneutrophil infiltrate. Patients receive active α₁PI with a target bloodthreshold of 35 μM active α₁PI and are monitored for active and inactiveα₁PI levels as well as for changes in the number of neutrophils incirculation using flow cytometry and changes in infection driveninflammation.

8. α₁PI augmentation therapy for neutropenia. In the majority ofpatients with severe congenital neutropenia, mutations are found in thegene encoding HLE or in the gene encoding the receptor for G-CSF(Horwitz et al., 1999; Benson et al., 2003). HLE mutations that preventlocalization to the plasma membrane cause cyclic neutropenia, andmutations that cause exclusive localization to the plasma membrane causethe pre-leukemic disorder, severe congenital neutropenia (Benson et al.,2003). Because inactive α₁PI mobilizes neutrophils (Joslin et al.,1992), augmentation with inactive α₁PI is used therapeutically for thepurpose of increasing the number of neutrophils in circulation. Patientsreceive inactive α₁PI with a target of 39 mg/kg inactive α₁PI and aremonitored for active and inactive α₁PI levels as well as for changes inthe number of neutrophils in circulation using flow cytometry.

9. α₁PI augmentation therapy for solid tumors. Tumor cell lines andbiopsy specimens exhibit inverse correlations of α₁PI and themetalloproteinase MMP-26 (Li et al., 2004). Expression of MMP-26 inestrogen-dependent neoplasms is likely to contribute to the inactivationof α₁PI promoting matrix destruction and malignant progression.Furthermore, evidence suggests α₁PI participates in tumor cell migration(Nejjari et al., 2004).

A serious side effect of myelosuppressive chemotherapy for solid tumors,is neutropenia. G-CSF (Filgrastim, Neupogen® or Neulasta®, Amgen, Inc.)is currently used to mobilize neutrophils in patients onmyelosuppressive chemotherapy. In combination with G-CSF, using activeα₁PI therapeutically to mobilize lymphoid-lineage cells in patientsreceiving myelosuppressive chemotherapy offers the additional benefit ofcontrolling tumor metastasis. Patients receive active α₁PI with a targetblood threshold of 35 μM active α₁PI and are monitored for active andinactive α₁PI levels as well as for changes in the number ofmyeloid-lineage, lymphoid-lineage, and tumor cells in circulation usingflow cytometry.

10. α₁PI augmentation therapy in atherosclerosis. Diminished active α₁PIpromotes atherogenesis (Talmud et al., 2003). Oxidized α₁PI has noproteinase inhibitory activity, and instead associates with LDL in vivo(Mashiba et al., 2001). The C-terminal fragment of α₁PI is present inatherosclerotic plaques (Dichtl et al., 2000). The oxidized andproteolyzed inactivation of α₁PI is thought to result from subclinicalinfections of the arterial intima by bacteria such as Porphyromonasgingivalis (Brodala et al., 2005; Beck et al., 2005) . Augmentation withactive α₁PI is used therapeutically to mobilize lymphoid-lineage cellsinto the infected tissue for the purpose of controlling and clearing theinfection. Patients receive augmentation with active α₁PI with a targetblood threshold of 35 μM active α₁PI and are monitored for active andinactive α₁PI levels as well as for intimal wall thickness andatherosclerotic plaque formation.

11. α₁PI augmentation therapy in insulin-dependent diabetes. Increasedinactive α₁PI is present in insulin-dependent diabetes due to thepresence of subclinical infections (Bristow et al., 1998; Sandler etal., 1988) and hyperglycemia (Sandler et al., 1988). Recombinantadeno-associated virus-mediated α₁PI gene therapy in a murine modelreduced the level of insulin autoantibodies and the frequency of overtdiabetes (Song et al., 2004). Augmentation with active α₁PI is usedtherapeutically to mobilize lymphoid-lineage cells into the infectedtissue for the purpose of controlling and clearing the infection as wellas to ameliorate the incidence of autoantibodies in diabetes. Patientsreceive augmentation with active α₁PI with a target blood threshold of35 μM active α₁PI and are monitored for active and inactive α₁PI levelsas well as for the presence of anti-insulin antibodies.

12. α₁PI augmentation therapy in autoimmune diseases. A predisposingcondition for the occurrence of autoimmune disease is the inborndeficiency of a proteinase or proteinase inhibitor involved inhomeostasis. Wegener's granulomatosis is caused by autoimmunity to theα₁PI ligand, proteinase 3 (Pendergraft et al., 2003; Csernok et al.,1990). Active α₁PI ameliorates the autoimmune pathogenesis of Wegener'sgranulomatosis (Rooney et al., 2001). Systemic lupus erythematosis canarise in patients with complement deficiencies or α₁PI deficiency(Sinico et al., 2005) Elevated HLE_(G) activity is detected in patientswith rheumatoid arthritis (Adeyemi et al., 1986). These patients benefitfrom augmentation with active α₁PI. Patients receive augmentation withactive α₁PI with a target blood threshold of 35 μM active α₁PI and aremonitored for active and inactive α₁PI levels as well as forautoimmune-mediated inflammation.

13. α₁PI augmentation therapy in solid organ transplantation. Excessiveactivation of proteinase cascade systems has been associated withpost-transplantation inflammatory disorders and organ rejection(Kirschfink, 2002). Augmentation with active α₁PI diminishespost-transplantation inflammation; however, this therapy also mobilizesboth lymphoid-lineage and myeloid-lineage cells potentially facilitatingorgan rejection. To overcome this adverse affect, α₁PI is geneticallymodified to prevent interaction with receptors and to preventstimulation of cell motility (see Example 5). Augmentation withgenetically modified α₁PI is used therapeutically to diminishinflammation and prevent recruitment of inflammatory blood cells andtheir products into transplants. Patients receive genetically modifiedα₁PI with a target blood threshold of 35 μM genetically modified α₁PIand are monitored for active and inactive α₁PI levels as well as formarkers of organ rejection.

14. α₁PI augmentation therapy in stem cell transplantation. Migration ofstem cells to, and progenitor cells from bone marrow is controlled byHLE_(CS), SDF-1, CXCR4 (Tavor. S. et al., 2005; Lapidot and Petit, 2002)and α₁PI (Examples 1-3 herein). Active α₁PI mobilizes lymphoid-lineagecells and inactive α₁PI mobilizes myeloid-lineage cells. Active andmodified α₁PI are used therapeutically to mobilize stem cells tohematopoietic tissue and progenitor cells from hematopoietic tissueduring stem cell transplantation.

Patients undergoing stem cell transplantation are treated with G-CSF(Filgrastim, Neupogen® or Neulasta®, Amgen, Inc.) to mobilize progenitorcells into circulation, and these are primarily myeloid-committedprogenitor cells (Cottler-Fox et al., 2003). Progenitor cells areharvested from blood and placed in culture in vitro for the purpose ofproliferation before transplantation. Proliferation and differentiationis monitored using flow cytometry. Active α₁PI is given therapeuticallywith a target blood threshold of 200_(R)M active α₁PI to mobilizelymphoid-lineage cells into circulation. Mobilized lymphoid-committedprogenitor cells are harvested from blood and placed in culture in vitrofor the purpose of proliferation before transplantation. Patientsreceiving mobilization treatment with active α₁PI are monitored foractive and inactive α₁PI levels. Harvested lymphoid-committed progenitorcells are monitored for proliferation and differentiation using flowcytometry prior to reinjection.

15. α₁PI in producing dendritic cell-based vaccines. Autologous stemcell transplantation involves a process of harvesting stem cells fromcirculation, culturing the cells in vitro to proliferate, andreinjection into the patient. This same principle is used to produceautologous dendritic cell-based vaccines. Dendritic cells are used as avector to deliver selected immunogens to the lymph nodes where theirinteraction with T lymphocytes initiates an immune response to theselected immunogen. Dendritic cell-based vaccines are currently beingused to induce immunity to tumor antigens (Schuler et al., 2003).Monocytic or lymphocytic cells are harvested from the blood of a patientwith cancer, for example, malignant melanoma. Harvested cells arecultured in vitro in the presence of a cocktail of cytokines includingG-CSF and GM-CSF that induces their differentiation into eithermonocyte-derived dendritic cells or plasmacytoid dendritic cellsdepending on the combination of cytokines used (Messmer et al., 2002).Dendritic cells are loaded with an antigen, for example melanomapeptide, and reinjected into the patient (Palucka et al., 2005; Schuleret al., 2003). Patients are monitored for the presence ofmelanoma-specific lymphocytes.

Active α₁PI is used to stimulate in vitro differentiation oflymphoid-lineage and myeloid-lineage blood cells into dendritic cells.Differentiation and function of dendritic cells is monitored using flowcytometry and cytokine secretion as described previously (Messmer etal., 2002). Dendritic cells are pulsed with antigen and reinjected intothe patient. Patients receiving α₁PI-induced dendritic cells aremonitored for the presence of immunogen-specific lymphocytes.

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The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will be apparent tothose skilled in the art from the foregoing description and theaccompanying figures.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

In the Specification:

Please amend the specification as shown:

Please insert the following on page 1, after the priority paragraph:

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 15, 2012, isnamed 86519CON.txt and is 11,216 bytes in size.

Please delete the paragraph on page 8, lines 3-18 and replace it withthe following paragraph:

FIG. 3( a-d). Corresponding conformation at the 3F5-recognized domainsin α₁PI and CD4-complexed HIV-1 gp120. Structures for human α₁PI (1HP7)and CD4-complexed HIV-1 gp120 (1RZJ) from the NCBI Molecular ModelingDatabase (MMDB) were analyzed using Cn3D software(www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). Small carbohydratestructures were already associated with 1RZJ in MMDB, and the threeassociated with 1HP7 were added using Adobe Photoshop. HIV-1 gp120 isdepicted from two perspectives (a,b) with two α-helices highlighted (aa21-39 and 306-313). The gp120 peptide immunogen used to raise 1C1 and3F5 (aa 300-321) is located at the C-terminus of gp120, and the linearsegment YKVV (aa 315-318, SEO ID NO: 1) along with the M-17 and theoligosaccharide-linked NGT (aa 92-94), are within 8A° of theconformational epitope. The gp120-homologous domain in α₁PI is alsolocated at the C-terminus of the protein, and is depicted from twoperspectives (c,d) with the highlighted antiparallel β-sheet strand atthe base of the cleft (aa 369-389), as well as the α-helices that formthe mouth of the cleft (aa 28-44 and 259-277). M-385, whichdistinguishes human from chimpanzee α₁PI, is indicated along with GKVV(aa 386-389, SEO ID NO: 2), the oligosaccharide, andoligosaccharide-linked NST (aa 46-48). The proteinase reactive siteM-358, is indicated for orientation.

Please delete the paragraph on page 11, line 22 to page 12, line 1 andreplace it with the following paragraph:

3.1 Structural features of α₁PI: The following represents the fulllength amino acid sequence for α₁PI (accession # K01396) including the24 aa signal peptide (SEO ID NO: 3):

−24 MPSSVSWGIL LLAGLCCLVP VSLA 1EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQS N STNI 51FFSPVSIATA FAMLSLGTKA DTHDEILEGL NF N LTEIPEA QIHEGFQELL 101RTLNQPDSQL QLTTGNGLFL SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT 151EEAKKQINDY VEKGTQGKIV DLVKELDRDT VFALVNYIFF KGKWERPFEV 201KDTEEEDFHV DQVTTVKVPM MKRLGMFNIQ HCKKLSSWVL LMKYLG N ATA 251IFFLPDEGKL QHLENELTHD IITKFLENED RRSASLHLPK LSITGTYDLK 301SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA 351MFLEAIPMSI PPEVKFNKPF VFLMIEQNTK SPLFMGKVVN PTQK

Please delete the paragraph on page 12, lines 20-22 and replace it withthe following paragraph: 3.2 Functional properties of α₁PI: There arethree distinct activities of α₁PI that are determined by sites in the C-terminal region of α₁PI defined herein as aa 357-394 (SEQ ID NO: 4),PMSI PPEVKFNKPF VFLMIEQNTK SPLFMGKVVN PTQK

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The first activity of α₁PI is its well characterized proteinaseinhibition which is a property only of active, uncleaved α₁PI. Thereactive site for this activity is Met (aa 358) contained in the domainPro-Met-Ser-Ile-Pro (PMSIP, aa 357-361, SEQ ID NO: 5). Active α₁PI maybe inactivated by proteinase complexing, cleavage, or oxidation of Met(aa 358). Interaction at the scissile bond Met-Ser (aa 358-359) may bemediated by many proteinases including HLE_(G). The two cleavageproducts of α₁PI may dissociate under some circumstances, but may remainassociated in a new, rearranged configuration that may irreversiblyincorporate HLE_(G), but may not incorporate other proteinases, forexample metalloproteinases (Perkins et al., 1992).

The tertiary structure for the rearranged α₁PI configuration has notbeen solved (Mellet et al., 1998); however, X-ray diffraction andkinetic analyses of cleaved α₁PI suggest that the strand SIPPEVKFNKP (aa359-369, SEQ ID NO: 6) may separate 70A° from its original position andinsert into the β-sheet formation on the opposite face of the molecule(β-sheet A) in a manner that would significantly alter proteinase andreceptor recognition (Elliott et al., 2000). Thus, four configurationsof the C-terminal region of α₁PI are thought to occur (Table 1).

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A second activity of α₁PI is the stimulation of cell migration, and thisactivity is a property of both cleaved and uncleaved α₁PI. Cleaved α₁PIis recognized by LRP, and stimulates migration of myeloid-lineage cellsincluding neutrophils and monocytic cells (Joslin et al., 1992). Active,uncleaved α₁PI is recognized by HLE_(CS) and stimulates migration oflymphoid-lineage cells and myeloid-committed progenitor cells (Bristowet al., 2003). Cell migration is initiated by α₁PI-induced co-capping ofreceptors such as HLE_(CS), CXCR4, and CD4 into podia formation(Cepinskas et al., 1999; Banda et al., 1988). In addition to theparticipation of podia formation during cell migration, thisconfiguration is also the preferred binding site for HIV-1 (Bristow etal., 2003). The reactive site in α₁PI for this activity isPhe-Val-Phe-Leu-M (FVFLM, aa 370-374, SEQ ID NO: 7).

A third non-physiologic activity of α₁PI is binding to antibodiesreactive with HIV-1 envelope protein gp120, and this activity results ininactivation of α₁PI and blocking of the other two activities describedabove. The anti-gp120 monoclonal antibodies 1C1 (Repligen, Inc.,Cambridge, Mass.) and 3F5 (hybridoma culture supernatant,0085-P3F5-D5-F8, Dr. Larry Arthur, NCI-Frederick) were previously shownto be reactive with an epitope near the gp120 C5 domain (Moore et al.,1994). The antibody cross-reactive site of human α₁PI is contained inthe domainPhe-Leu-Met-Ile-Glu-Gln-Asn-Thr-Lys-Ser-Pro-Leu-Phe-Met-Gly-Lys-Val-Val(FLMIEQNTKSPLFMGKVV, aa 372-389, SEQ ID NO: 8) (Bristow et al., 2001)Chimpanzee α₁PI, which differs from human α₁PI by a single amino acid,Val (aa 385), does not bind anti-gp120, consistent with the ability ofchimpanzees to resolve HIV-1 infection and regain normal CD4⁺ lymphocytelevels. This suggests that the anti-gp120 cross-reactive site in humanα₁PI is determined primarily by the Met residue (aa 385).

3.3 Expression of recombinant α₁PI: Any method known in the art may beused for producing genetically modified α₁PI's according to theinvention. Two preferred methods are briefly described below forproducing such recombinant α₁PI's; one method allows expression of α₁PIin rice cells and the other allows bacterial expression. The cDNAencoding human α₁PI is obtained from a human cDNA bank by andamplification of the fragment in accession number K01396 using two PCRprimers: N-terminal primer 5′ GAGGATCCCCAGGGAGATGCTGCCCAGAA 3′ (SEQ IDNO: 9) and C-terminal primer 5′CGCGCTCGAGTTATTTTTGGGTGGGATTCACCAC 3′(SEQ ID NO: 10) as previously described (Courtney et al., 1984;Terashima et al., 1999; Jean et al., 1998).

For expression in rice cells, expression cassettes are prepared by usinga 1.1 kb NheI-PstI fragment, derived from p1AS1.5, is cloned into thevector pGEM5zf- (Promega, Madison, Wis.): ApaI, AatII, SphI, Ncol,SstII, EcoRV, SpeI, NotI, PstI, SalI, NM, SacI, MluI, NsiI at the SpeIand PstI sites to form pGEM5zf-(3D/NheI-PstI). The GEM5zf-(3D/NheI-PstI)is digested with PstI and Sad and ligated in two nonkinased 30mers withthe complementary sequences 5′ GCTTG ACCTG TAACT CGGGC CAGGC GAGCT 3 ‘(SEQ ID NO: 11) and 5’ CGCCT AGCCC GAGTT ACAGG TCAAG CAGCT 3′ (SEQ IDNO: 12) to form p3DProSig. A 5-kb BamHI-Kpnl fragment from lambda clone□OSgl A is used as a terminator. Hygromycin resistance is obtained fromthe 3-kb BamHI fragment containing the 35S promoter-Hph-NOS of theplasmid pMON410.

Please delete the paragraphs on page 16, line 11 to page 17, line 4 andreplace them with the following paragraphs:

The genetic modifications of interest are described in Section 4.1 ofthe Detailed Description. Site-directed mutagenesis of active α1PI isperformed using standard procedures which are well known in the art(e.g., Parfrey et al., 2003; Current Protocols in Molecular Biology,2002). For example, the DNA sequence encoding the human α1PI signalpeptide in pDS56α₁PI/hf is replaced with sequences encoding the epitope(FLAG)-tag by insertion of the annealed complimentary oligos 5′CTAGAGGATCCCATGGACTACAAGGACGACGATGACAAGGAA 3′ (SEQ ID NO: 13) and5′GATCTTCCTTGTCATCGTCGTCCTTGTAGTCCATGGGATCCT 3′ (SEQ ID NO: 14). Theresulting cDNA is subcloned into pDS56-6His to generate pDS56α1PI/hf. Togenerate pDS56α1PI/hf carrying an amino acid substitution, the DNAsequences encoding the wild-type amino acid are replaced by thecomplimentary oligos coding for the amino acids described in Section 4.1of the Detailed Description. The resulting ORFs directed cytosolicexpression of the recombinant proteins initiating with a Met followed bythe His and FLAG tags and the mature sequences of mutant α1PI.

4.1 Genetic modification within the domain that determines cellmigration (FVFLM, aa 370-374, SEQ ID NO: 7) is prepared by site-directedmutagenesis of specific amino acids:

4.1.1 Phe (aa 370) to Ile, Leu, Val, Tyr, or Gly.

4.1.2 Val (aa 371) to Phe, Leu, Ile, or Gly.

4.1.3 Phe (aa 372) to Ile, Leu, Val, Tyr or Gly.

4.1.4 Leu (aa 373) to Ile, Val, Phe, or Gly.

-   -   4.1.5 Met (aa 374) to Phe, Thr, Ile, Leu, Val, or Gly.

Please delete the paragraph on page 17, line 23 to page 18, line 4 andreplace it with the following paragraph:

The C-terminal α₁PI proteolytic fragments acquire attributes that allowinteraction with the LDL receptor-related protein (LRP) (Poller et al.,1995) and other receptors that recognize a pentapeptide sequence FVFLM(aa 370-374, SEQ ID NO: 7) (Joslin et al., 1992) in a manner thatproduces, chemotaxis of neutrophils, increased LDL binding to monocytes,upregulated LDL receptors, increased cytokine production, and α₁PIsynthesis (Banda et al., 1988; Janciauskiene et al., 1999; Janciauskieneet al., 1999). It has been shown that fibrillar aggregates of theC-terminal fragment of α₁PI facilitate uptake of LDL by LRP on thehepatolastoma cell line HepG2 (Janciauskiene and Lindgren, 1999), andthese fragments participate in atherosclerosis (Dichtl et al., 2000).

Please delete the paragraphs on page 25, line 13 to page 26, line 9 andreplace them with the following paragraphs:

The gp120 epitope recognized by 1C1 and 3F5 is considered to beconformation-dependent (Moore et al., 1994). The gp120 peptide immunogenused to raise 1C1 and 3F5 (aa 300-321, GGGDMRDNWRSELYKYKVVK (SEQ ID NO:15) (Ratner et al., 1985) contains both an α-helix (aa 306-313) andlinear strand (aa 314-321) (FIG. 3 a,b), but other epitope determinantsof the antibodies are not known. In human α₁PI (FIG. 3 c,d), thegp120-homologous sequence (aa 369-389, PFVFLMIDQNTKSPLFMGKVV (SEQ ID NO:16) folds to form a two-stranded antiparallel β-sheet that lies at thebase of a cleft (4A° deep by 20A° long by 5A° wide) topped by twoα-helices (aa 28-47 and 259-277) in a smaller, but similar configurationas the antigen-binding cleft of MHC (10A° deep by 25A° long by 10A°wide) (Bjorkman et al., 1987). At the far end of the first of theseα-helices is the N-linked mannose-containing oligosaccharide thatconfers structural polymorphism to α₁PI (aa 46) (Jeppsson et al., 1985).In the center of the α-sheet that lies in the cleft, is M-385 whichdistinguishes human from chimpanzee α₁PI (V-385): The function of thiscleft is not known, but a sequence in the center of the β-sheetformation (aa 370-374, FVFLM (SEQ ID NO: 7) is homologous to the fusiondomain of HIV-1 gp41, and this sequence has been implicated in bindingHLE_(CS) (Bristow et al., 1995; Bristow et al., 2003) and stimulatingcell motility (Joslin et al., 1991).

In α₁PI, the sequence GKVV (aa 386-389, SEQ ID NO: 2) lies within 5A° ofM-385, N-46, and the N-linked oligosaccharide in a space occupying 5A°by 5A° by 5A° . In gp120, in the same relative orientation as in α₁PI,the sequence YKVV (aa 315-318, SEQ ID NO: 1) lies within 5A° of M-17 and8A° of N-92 and the N-linked mannose-containing oligosaccharide (Leonardet al., 1987) in a space occupying 5A° by 5A° by 8A° . Evidence thatα₁PI polymorphisms may influence 3F5 binding (FIG. 2 a) suggests thepolymorphism-determining N-46 oligosaccharide participates in 3F5recognition of α₁PI. Thus, the 3F5 conformational epitope is suggestedby these analyses to occupy a 5A° by 5A° by 8A° space including KVV, M,N, and the N-linked oligosaccharide. This proposed conformationalepitope is consistent with previously characterized antigenconfigurations that contain oligosaccharide determinants (Cygler et al.,1991) as well as with results demonstrating that gp120 N-92 is invariant(Wei et al., 2003).

Please delete the paragraph on page 28, lines 8-13 and replace it withthe following paragraph: α₁PI is genetically modified as described inSection 4 of the Detailed Description with three substitutions, aa 385(Met to Val), aa 372 (Phe to Gly), and aa 373 (Leu to Gly), and isdesignated α₁PI.β.F372G.L373G.M385V (α₁PI.β. The α₁PI.β sequence with aachanges represented in bold underlined letters is as follows (SEQ ID NO:17):

−24 MPSSVSWGIL LLAGLCCLVP VSLA 1EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI 51FFSPVSIATA FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL 101RTLNQPDSQL QLTTGNGLFL SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT 151EEAKKQINDY VEKGTQGKIV DLVKELDRDT VFALVNYIFF KGKWERPFEV 201KDTEEEDFHV DQVTTVKVPM MKRLGMFNIQ HCKKLSSWVL LMKYLGNATA 251IFFLPDEGKL QHLENELTHD IITKFLENED RRSASLHLPK LSITGTYDLK 301SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA 351MFLEAIPMSI PPEVKFNKPF V GG MIEQNTK SPLF V GKVVN PTQK

1. A method for identifying a modified alproteinase inhibitor assuitable for use in treating a disease, disorder or condition in asubject, comprising: (a) producing the modified alproteinase inhibitor;and (b) measuring a biological activity of the modified alproteinaseinhibitor in a biological assay for predicting effectiveness in treatingthe disease, disorder or condition in the subject, wherein the modifiedalproteinase inhibitor is identified as suitable for treating thedisease, disorder or condition from a change in the biological activityrelative to a control activity measured for a wild-type alproteinaseinhibitor.
 2. The method of claim 1, wherein the modified alproteinaseinhibitor is produced by site-directed mutagenesis, proteolysis, orboth.
 3. The method of claim 1, wherein the disease, disorder orcondition is selected from the group consisting of HIV-1 infection,bacterial infection, leukemia, a solid tumor, atherosclerosis, anautoimmune disease, organ transplantation, and stem celltransplantation.
 4. The method of claim 1, wherein the biological assayis selected from the group consisting of an elastase inhibition assay, areceptor co-capping assay, a cell motility assay, a lymphoid-committedprogenitor cell mobilization assay, an HIV-1 gp120 antibodycross-reactivity assay, and an HIV-1 infectivity facilitation assay. 5.The method of claim 1, wherein the subject is a human or a non-humananimal.
 6. The method of claim 2, wherein proteolysis comprisescontacting a wild-type or a recombinant alproteinase inhibitor with aprotease selected from the group consisting of elastase, stromelysin-3,matrix metalloproteinase, collagenase, gelatinase, pepsin, plasmin,urokinase, chymotrypsin, thrombin, CD26, complement component Cl, andcomplement component C3.
 7. The method of claim 2, wherein site-directedmutagenesis comprises changing a wild-type amino acid selected from thegroup consisting of residues 370-374 and 385 to a non-wild-type residue.8. The method of claim 7, wherein at least one amino acid selected fromthe group consisting of residues 370-374 and 385 is changed fromwild-type to glycine, threonine, or a hydrophobic amino acid.
 9. Themethod of claim 8, wherein the hydrophobic amino acid is selected fromthe group consisting of isoleucine, leucine, phenylalanine, tyrosine andvaline.
 10. A modified human alproteinase inhibitor comprising a changein a wild-type amino acid residue selected from the group consisting ofresidues 370-374 and
 385. 11. The modified human alproteinase inhibitorof claim 10, wherein the modification further comprises proteolysis. 12.The modified human alproteinase inhibitor of claim 10, wherein thechange is to glycine, threonine, or a hydrophobic amino acid.
 13. Themodified human alproteinase inhibitor of claim 12, wherein thehydrophobic amino acid is selected from the group consisting ofisoleucine, leucine, phenylalanine, tyrosine and valine.
 14. Themodified human alproteinase inhibitor of claim 10, which comprises atleast two changes in wild-type amino acid residues selected from thegroup consisting of residues 370-374 and
 385. 15. The modified humanalproteinase inhibitor of claim 14, wherein methionine at position 385is changed to a non-methionine amino acid.
 16. The modified humanalproteinase inhibitor of claim 15, wherein the non-methionine aminoacid is selected from the group consisting of glycine, isoleucine,leucine, phenylalanine, threonine, and valine.
 17. The modified humanalproteinase inhibitor of claim 16 which is capable of a reduced bindingactivity in an HIV-1 gp120 antibody cross-reactivity assay, relative toa wild-type alproteinase inhibitor.
 18. The modified human alproteinaseinhibitor of claim 10 comprising the following three amino acidsubstitutions: Phe372Gly; Leu373G1y; and Met 385Val.
 19. The modifiedhuman alproteinase inhibitor of claim 10 consisting of the followingthree amino acid substitutions: Phe372Gly; Leu373Gly; and Met 385Val.20. A method of treating a disease, disorder or condition in a subjectin need of said treatment, comprising administering an amount effectiveto treat said disease disorder or condition of a modified alproteinaseinhibitor to the subject.
 21. The method of claim 20, wherein themodified alproteinase inhibitor is produced by site-directedmutagenesis, proteolysis, or both.
 22. The method of claim 20, whereinthe disease, disorder or condition is selected from the group consistingof HIV-1 infection, bacterial infection, leukemia, a solid tumor,atherosclerosis, an autoimmune disease, organ transplantation, and stemcell transplantation.
 23. The method of claim 20, wherein the subject isa human or a non-human animal.
 24. The method of claim 20, wherein themodified alproteinase inhibitor comprising a change in a wild-type aminoacid residue selected from the group consisting of residues 370-374 and385.
 25. The method of claim 24, wherein wild-type methionine atposition 385 is changed to a non-methionine amino acid, and wherein thenon-methionine amino acid is selected from the group consisting ofglycine, isoleucine, leucine, phenylalanine, threonine, and valine. 26.The method of claim 25, wherein the modified alproteinase inhibitor iscapable of a reduced binding activity in an HIV-1 gp120 antibodycross-reactivity assay, relative to a wild-type alproteinase inhibitor.27. The method of claim 25, wherein the modified alproteinase inhibitorcomprises the following three amino acid substitutions: Phe372Gly;Leu373Gly; and Met 385Val.
 28. The method of claim 25, wherein themodified alproteinase inhibitor consists of the following three aminoacid substitutions: Phe372Gly; Leu373Gly; and Met 385Val.
 29. The methodof claim 27 or 28, further comprising administration of antiretroviraltherapy.
 30. The method of claim 20, wherein the effective amount ofmodified alproteinase inhibitor is a dose equivalent to about 42 mg/kgof active wild-type alproteinase inhibitor.
 31. The method of claim 3,wherein stem cell transplantation is autologous.
 32. A method oftreating a disease, disorder or condition in a subject in need of saidtreatment, comprising administering an amount effective to treat saiddisease-disorder or condition of an active alproteinase inhibitor to thesubject, wherein the disease, disorder or condition is selected from thegroup consisting of HIV-1 infection, bacterial infection, leukemia, asolid tumor, atherosclerosis, an autoimmune disease, organtransplantation, and stem cell transplantation.
 33. The method of claim32, wherein stem cell transplantation is autologous.
 34. A method oftreating a disease, disorder or condition in a subject in need of saidtreatment, comprising administering an amount effective to treat saiddisease disorder or condition of an active alproteinase inhibitor to thesubject, wherein the subject is characterized as having an abnormalnumber of lymphocytes, monocytes, or dendritic cells.
 35. A method oftreating a disease, disorder or condition in a subject in need of saidtreatment, comprising administering an amount effective to treat saiddisease disorder or condition of an inactive alproteinase inhibitor tothe subject, wherein the disease, disorder or condition is selected fromthe group consisting of bacterial infection, neutropenia andimmunosuppression.
 36. A method of treating a disease, disorder orcondition in a subject in need of said treatment, comprisingadministering an amount effective to treat said disease disorder orcondition of an inactive alproteinase inhibitor to the subject, whereinthe subject is characterized as having an abnormal number ofgranulocytes, monocytes, dendritic cells, eosinophils, or basophils. 37.The method of claim 32-35 or 36, wherein the subject is a human or anon-human animal.
 38. A method for treating a subject suffering fromabnormalities in the number of cells of myeloid or lymphoid lineage thatare associated with HIV-1 infection, microbial infection, leukemia,solid tumor cancers, atherosclerosis, autoimmunity, stem celltransplantation or organ transplantation comprising administering to asubject in need of such treatment an amount effective to treat saidsubject of an alproteinase inhibitor.
 39. The method of claim 38 whereinsaid subject is a human or a non-human or animal.
 40. The method ofclaim 38, wherein said stem cell transplantation is autologous.